ABSTRACT
This corrects the article DOI: 10.1038/mi.2017.68.
ABSTRACT
We previously demonstrated that protein kinase C-δ (PKCδ) is critical for immunity against Listeria monocytogenes, Leishmania major, and Candida albicans infection in mice. However, the functional relevance of PKCδ during Mycobacterium tuberculosis (Mtb) infection is unknown. PKCδ was significantly upregulated in whole blood of patients with active tuberculosis (TB) disease. Lung proteomics further revealed that PKCδ was highly abundant in the necrotic and cavitory regions of TB granulomas in multidrug-resistant human participants. In murine Mtb infection studies, PKCδ-/- mice were highly susceptible to tuberculosis with increased mortality, weight loss, exacerbated lung pathology, uncontrolled proinflammatory cytokine responses, and increased mycobacterial burdens. Moreover, these mice displayed a significant reduction in alveolar macrophages, dendritic cells, and decreased accumulation of lipid bodies (lungs and macrophages) and serum fatty acids. Furthermore, a peptide inhibitor of PKCδ in wild-type mice mirrored lung inflammation identical to infected PKCδ-/- mice. Mechanistically, increased bacterial growth in macrophages from PKCδ-/- mice was associated with a decline in killing effector functions independent of phagosome maturation and autophagy. Taken together, these data suggest that PKCδ is a marker of inflammation during active TB disease in humans and required for optimal macrophage killing effector functions and host protection during Mtb infection in mice.
Subject(s)
Biomarkers/metabolism , Granuloma, Respiratory Tract/immunology , Lung/immunology , Macrophages/immunology , Mycobacterium tuberculosis/physiology , Protein Kinase C-delta/metabolism , Tuberculosis, Pulmonary/immunology , Adolescent , Adult , Animals , Cohort Studies , Cross-Sectional Studies , Cytotoxicity, Immunologic , Female , Granuloma, Respiratory Tract/microbiology , Humans , Macrophages/microbiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Kinase C-delta/genetics , ProteomicsABSTRACT
Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFß1 mRNA, and addition of exogenous TGFß1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFßR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFß signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.
Subject(s)
Lung Neoplasms/genetics , Protein Kinase C-alpha/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Signal Transduction/genetics , Transforming Growth Factor beta/genetics , p38 Mitogen-Activated Protein Kinases/genetics , Animals , Bronchioles/metabolism , Bronchioles/pathology , Cells, Cultured , Disease Models, Animal , Enzyme Activation , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Inhibitor of Differentiation Proteins/genetics , Inhibitor of Differentiation Proteins/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Protein Kinase C-alpha/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Pulmonary Alveoli/metabolism , Pulmonary Alveoli/pathology , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/metabolism , Stem Cells/pathology , Transforming Growth Factor beta/metabolism , WT1 Proteins/genetics , WT1 Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
AIMS/HYPOTHESIS: Protein kinase Cε (PKCε) is emerging as a key mediator of lipid-induced insulin resistance in liver and hepatic lipid metabolism itself. We investigated whether PKCε plays a role in other metabolic processes, to further examine its suitability as a therapeutic target. METHODS: We measured amino acid, organic acid and sugar levels by liquid and gas chromatography-mass spectrometry of liver extracts from chow and fat-fed wild-type (WT) and PKCε-deficient (Prkce(-/-)) mice. Fed and fasting glucose, ketone and fatty acid levels were measured in blood. Triacylglycerol levels and gluconeogenic and ketogenic enzyme expression were measured in liver. The effect of fasting on epididymal fat pad mass was also determined. RESULTS: Metabolomic analysis indicated that the short-term high-fat diet affected over 20 compounds, including a 50% reduction in the glucogenic amino acid alanine. Prkce deletion resulted only in a reduction of 4-hydroxyproline and aspartate and an increase in glutamate. However, upon fasting, Prkce(-/-) mice were better able to maintain blood glucose levels and also exhibited lower levels of the ketone ß-hydroxybutyrate compared with WT mice. Upon fasting, Prkce deletion also resulted in lower liver and plasma lipids and a smaller reduction in fat pad mass. CONCLUSIONS/INTERPRETATION: Metabolomic analysis provided new insights into the effects of a high-fat diet on liver metabolite levels. Glucose homeostasis under fasting conditions is improved in Prkce(-/-) mice, which, in turn, may reduce the mobilisation of lipid from adipose tissue, reducing the availability of ketogenic substrate in the liver. Together with the protection against fat-diet-induced glucose intolerance previously observed in the fed state, these findings indicate PKCε as a unique therapeutic target for the improvement of glucose homeostasis.
Subject(s)
Fasting/metabolism , Gluconeogenesis/physiology , Ketones/metabolism , Liver/metabolism , Protein Kinase C-epsilon/deficiency , Animals , Fatty Acids/metabolism , Gene Deletion , Hemostasis/physiology , Insulin Resistance/physiology , Mice , Mice, Knockout , Models, Animal , Protein Kinase C-epsilon/genetics , Protein Kinase C-epsilon/physiologyABSTRACT
AIMS/HYPOTHESIS: We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice. METHODS: Prkce(-/-) and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes. RESULTS: While fat-fed WT mice became glucose intolerant after 1 week, Prkce(-/-) mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce(-/-) animals. Liver triacylglycerols were increased in fat-fed Prkce(-/-) mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce(-/-) mice. Prkce(-/-) hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce(-/-) mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported. CONCLUSIONS/INTERPRETATION: Prkce deletion ameliorates diet-induced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails.
Subject(s)
Dietary Fats/metabolism , Glucose/metabolism , Homeostasis/physiology , Lipid Metabolism/physiology , Liver/metabolism , Protein Kinase C-epsilon/deficiency , Animals , Gene Deletion , Insulin/metabolism , Mice , Mice, Knockout , Models, Animal , Protein Kinase C-epsilon/genetics , Reactive Oxygen Species/metabolism , Time FactorsABSTRACT
AIMS/HYPOTHESIS: Proinflammatory cytokines contribute to beta cell destruction in type 1 diabetes, but the mechanisms are incompletely understood. The aim of the current study was to address the role of the protein kinase C (PKC) isoform PKCδ, a diverse regulator of cell death, in cytokine-stimulated apoptosis in primary beta cells. METHODS: Islets isolated from wild-type or Prkcd(-/-) mice were treated with IL-1ß, TNF-α and IFNγ and assayed for apoptosis, nitric oxide (NO) generation and insulin secretion. Activation of signalling pathways, apoptosis and endoplasmic reticulum (ER) stress were determined by immunoblotting. Stabilisation of mRNA transcripts was measured by RT-PCR following transcriptional arrest. Mice were injected with multiple low doses of streptozotocin (MLD-STZ) and fasting blood glucose monitored. RESULTS: Deletion of Prkcd inhibited apoptosis and NO generation in islets stimulated ex vivo with cytokines. It also delayed the onset of hyperglycaemia in MLD-STZ-treated mice. Activation of ERK, p38, JNK, AKT1, the ER stress markers DDIT3 and phospho-EIF2α and the intrinsic apoptotic markers BCL2 and MCL1 was not different between genotypes. However, deletion of Prkcd destabilised mRNA transcripts for Nos2, and for multiple components of the toll-like receptor 2 (TLR2) signalling complex, which resulted in disrupted TLR2 signalling. CONCLUSIONS/INTERPRETATION: Loss of PKCδ partially protects against hyperglycaemia in the MLD-STZ model in vivo, and against cytokine-mediated apoptosis in vitro. This is accompanied by reduced NO generation and destabilisation of Nos2 and components of the TLR2 signalling pathway. The results highlight a mechanism for regulating proinflammatory gene expression in beta cells independently of transcription.
Subject(s)
Apoptosis/drug effects , Cytokines/pharmacology , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Isoenzymes/metabolism , Protein Kinase C-delta/metabolism , Animals , Apoptosis/genetics , Blotting, Western , In Vitro Techniques , Insulin-Secreting Cells/drug effects , Interferon-gamma/pharmacology , Interleukin-1beta/pharmacology , Islets of Langerhans/cytology , Isoenzymes/genetics , Mice , Mice, Knockout , Phosphorylation/drug effects , Polymerase Chain Reaction , Protein Kinase C-delta/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Activators of 5'-AMP-activated protein kinase (AMPK) 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), metformin, and exercise activate atypical protein kinase C (aPKC) and ERK and stimulate glucose transport in muscle by uncertain mechanisms. Here, in cultured L6 myotubes: AICAR- and metformin-induced activation of AMPK was required for activation of aPKC and ERK; aPKC activation involved and required phosphoinositide-dependent kinase 1 (PDK1) phosphorylation of Thr410-PKC-zeta; aPKC Thr410 phosphorylation and activation also required MEK1-dependent ERK; and glucose transport effects of AICAR and metformin were inhibited by expression of dominant-negative AMPK, kinase-inactive PDK1, MEK1 inhibitors, kinase-inactive PKC-zeta, and RNA interference (RNAi)-mediated knockdown of PKC-zeta. In mice, muscle-specific aPKC (PKC-lambda) depletion by conditional gene targeting impaired AICAR-stimulated glucose disposal and stimulatory effects of both AICAR and metformin on 2-deoxyglucose/glucose uptake in muscle in vivo and AICAR stimulation of 2-[(3)H]deoxyglucose uptake in isolated extensor digitorum longus muscle; however, AMPK activation was unimpaired. In marked contrast to AICAR and metformin, treadmill exercise-induced stimulation of 2-deoxyglucose/glucose uptake was not inhibited in aPKC-knockout mice. Finally, in intact rodents, AICAR and metformin activated aPKC in muscle, but not in liver, despite activating AMPK in both tissues. The findings demonstrate that in muscle AICAR and metformin activate aPKC via sequential activation of AMPK, ERK, and PDK1 and the AMPK/ERK/PDK1/aPKC pathway is required for metformin- and AICAR-stimulated increases in glucose transport. On the other hand, although aPKC is activated by treadmill exercise, this activation is not required for exercise-induced increases in glucose transport, and therefore may be a redundant mechanism.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Enzyme Activators/pharmacology , Glucose Transport Proteins, Facilitative/metabolism , Metformin/pharmacology , Muscle Fibers, Skeletal/drug effects , Protein Kinase C/metabolism , Ribonucleosides/pharmacology , AMP-Activated Protein Kinases/drug effects , AMP-Activated Protein Kinases/metabolism , Aminoimidazole Carboxamide/pharmacology , Animals , Blood Glucose/metabolism , Enzyme Activation/drug effects , Enzyme Activation/physiology , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Glucose Transport Proteins, Facilitative/drug effects , Hypoglycemic Agents/pharmacology , In Vitro Techniques , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mice , Mice, Knockout , Muscle Fibers, Skeletal/metabolism , Physical Conditioning, Animal/physiology , Protein Kinase C/drug effects , Protein Kinase C/genetics , Rats , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
AIMS/HYPOTHESIS: This study aimed to determine whether protein kinase C (PKC) delta plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCepsilon in the generation of insulin resistance in skeletal muscle. METHODS: Prkcd (-/-), Prkce (-/-) and wild-type mice were fed high-fat diets and subjected to glucose tolerance tests. Blood glucose levels and insulin responses were determined during the tests. Insulin signalling in liver and muscle was assessed after acute in vivo insulin stimulation by immunoblotting with phospho-specific antibodies. Activation of PKC isoforms in muscle from Prkce (-/-) mice was assessed by determining intracellular distribution. Tissues and plasma were assayed for triacylglycerol accumulation, and hepatic production of lipogenic enzymes was determined by immunoblotting. RESULTS: Both Prkcd (-/-) and Prkce (-/-) mice were protected against high-fat-diet-induced glucose intolerance. In Prkce (-/-) mice this was mediated through enhanced insulin availability, while in Prkcd (-/-) mice the reversal occurred in the absence of elevated insulin. Neither the high-fat diet nor Prkcd deletion affected maximal insulin signalling. The activation of PKCdelta in muscle from fat-fed mice was enhanced by Prkce deletion. PKCdelta-deficient mice exhibited reduced liver triacylglycerol accumulation and diminished production of lipogenic enzymes. CONCLUSIONS/INTERPRETATION: Deletion of genes encoding isoforms of PKC can improve glucose intolerance, either by enhancing insulin availability in the case of Prkce, or by reducing lipid accumulation in the case of Prkcd. The absence of PKCepsilon in muscle may be compensated by increased activation of PKCdelta in fat-fed mice, suggesting that an additional role for PKCepsilon in this tissue is masked.
Subject(s)
Dietary Fats/adverse effects , Glucose Intolerance/chemically induced , Protein Kinase C-delta/deficiency , Protein Kinase C-delta/metabolism , Protein Kinase C-epsilon/deficiency , Protein Kinase C-epsilon/metabolism , Animals , Blood Glucose/metabolism , Crosses, Genetic , Gene Deletion , Glucose Intolerance/blood , Glucose Intolerance/enzymology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Muscle, Skeletal/enzymology , Protein Kinase C-delta/genetics , Protein Kinase C-epsilon/genetics , Triglycerides/metabolismABSTRACT
The aim of our group is to identify PKC (protein kinase C) in vivo function by analysing individual PKC knockouts we have generated over the past few years. The general approach we are using to identify target tissues and/or defined cell populations within the mouse for further investigation is a detailed expression analysis of individual PKC isoforms. For these purposes, we have established several specific tools in the past that allow us to follow up isoform-specific PKC expression on a very precise level. Doing so, we have started to investigate PKC expression profiles under various tumour conditions in mice. As predicted, we were able to identify various PKC isoforms to be either up- or down-regulated during the development and progression of certain tumours, implying that these isoforms are substantially linked to the biology of these tumours. In order to prove this hypothesis, we then crossed relevant PKC knockout lines on the appropriate tumour background and analysed tumour growth and progression under PKC-deficient conditions. Exemplary of this approach, recent data generated with PKCalpha-deficient APC(Min) (adenomatous polyposis coli) mice identify PKCalpha in this system acting as a tumour suppressor instead of being a promoter as suggested from PMA data.
Subject(s)
Models, Animal , Protein Kinase C/metabolism , Animals , Gene Expression Profiling , Mice , Mice, Knockout , Protein Kinase C/geneticsABSTRACT
Albuminuria in diabetic nephropathy is due to endothelial dysfunction, a loss of negative charges in the basement membrane, and changes a of the slit-membrane diaphragm composition. We have recently shown that protein kinase C alpha (PKCalpha)-deficient mice are protected against the development of albuminuria under diabetic conditions. We here tested the hypothesis that PKCalpha mediates the hyperglycemia-induced downregulation of the slit-diaphragm protein nephrin. After 8 weeks of streptozotocin (STZ)-induced hyperglycemia the expression of glomerular nephrin was significantly reduced. In contrast, other slit-diaphragm proteins such as podocin and CD2AP were unaltered in diabetic state. In PKCalpha-/- mice, hyperglycemia-induced downregulation of nephrin was prevented. Podocin and CD2AP remained unchanged. In addition, the nephrin messenger RNA expression was also reduced in hyperglycemic wild-type mice but remained unaltered in PKCalpha-/- mice. We postulate that the underlying mechanism of the hyperglycemia-induced regulation of various proteins of the glomerular filtration barrier is a PKCalpha-dependent regulation of the Wilms' Tumor Suppressor (WT1) which previously has been shown to act as a direct transcription factor on the nephrin promoter. Our data suggest that PKCalpha activation may be an important intracellular signaling pathway in the regulation of nephrin expression and glomerular albumin permeability in the diabetic state.
Subject(s)
Diabetic Nephropathies/metabolism , Membrane Proteins/metabolism , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Signal Transduction/physiology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Albuminuria/etiology , Albuminuria/metabolism , Animals , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/physiopathology , Diabetic Nephropathies/physiopathology , Gene Expression Regulation/physiology , Humans , Hyperglycemia , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Kidney Glomerulus/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , WT1 Proteins/genetics , WT1 Proteins/metabolismABSTRACT
Smooth muscle cell (SMC) accumulation is a key event in the development of atherosclerosis, including vein bypass graft arteriosclerosis. Because members of the protein kinase C (PKC) family signal cells to undergo proliferation, differentiation, or apoptosis, we generated PKCdelta knockout mice and performed vein bypass grafts on these animals. PKCdelta(-/-) mice developed normally and were fertile. Vein segments from PKCdelta(-/-) mice isografted to carotid arteries of recipient mice of either genotype led to a more severe arteriosclerosis than was seen with PKCdelta(+/+) vein grafts. Arteriosclerotic lesions in PKCdelta(-/-) mice showed a significantly higher number of SMCs than were found in wild-type animals; this was correlated with decreased SMC death in lesions of PKCdelta(-/-) mice. SMCs derived from PKCdelta(-/-) aortae were resistant to cell death induced by any of several stimuli, but they were similar to wild-type SMCs with respect to mitogen-stimulated cell proliferation in vitro. Furthermore, pro-apoptotic treatments led to diminished caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release in PKCdelta(-/-) relative to wild-type SMCs, suggesting that their apoptotic resistance involves the loss of free radical generation and mitochondrial dysfunction in response to stress stimuli. Our data indicate that PKCdelta maintains SMC homeostasis and that its function in the vessel wall per se is crucial in the development of vein graft arteriosclerosis.
Subject(s)
Arteriosclerosis/genetics , Blood Vessel Prosthesis , Isoenzymes/metabolism , Protein Kinase C/metabolism , Veins/pathology , Animals , Arteriosclerosis/enzymology , Isoenzymes/genetics , Mice , Mice, Knockout , Protein Kinase C/genetics , Protein Kinase C-delta , Protein TransportABSTRACT
Here we have addressed the role that zetaPKC plays in NF-kappaB activation using mice in which this kinase was inactivated by homologous recombination. These mice, although grossly normal, showed phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-beta receptor gene-deficient mice. The lack of zetaPKC in embryonic fibroblasts (EFs) severely impairs kappaB-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of zetaPKC with p65 was detected which requires the previous degradation of IkappaB. Although in zetaPKC-/- EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses zetaPKC, IKK activation is inhibited.
Subject(s)
Acetylcysteine/analogs & derivatives , Fibroblasts/physiology , Gene Targeting , I-kappa B Proteins , NF-kappa B/metabolism , Protein Kinase C/genetics , Transcription, Genetic/genetics , Acetylcysteine/pharmacology , Animals , Apoptosis/physiology , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , Female , Fibroblasts/drug effects , Fibroblasts/enzymology , Genes, Reporter/genetics , I-kappa B Kinase , Interleukin-1/pharmacology , Lung/enzymology , Lung/physiology , Lymphocyte Subsets/metabolism , Male , Mice , Mice, Knockout , NF-KappaB Inhibitor alpha , Peyer's Patches/cytology , Peyer's Patches/metabolism , Phenotype , Phosphorylation , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Proteins/metabolism , Spleen/cytology , Spleen/metabolism , Transcription Factor RelA , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The understanding and control of many pathophysiological conditions is based on knowledge of subtly regulated intracellular signaling networks. We have found that in pervanadate (PV)-treated J558L myeloma cells, amongst other signaling proteins, protein kinase C (PKC)-delta and src homology 2-containing inositol phosphatase (SHIP) are tyrosine phosphorylated on expression of the B cell receptor, suggesting a role for these proteins in the preformed B cell receptor transducer complex. Rottlerin, a widely used PKC-delta-specific inhibitor, efficiently blocks these PV-induced tyrosine phosphorylation events. Furthermore, PV treatment of bone marrow-derived mast cells (BMMC) also results in tyrosine phosphorylation of PKC-delta, SHIP, and additional proteins. Rottlerin also inhibits these responses, indicating that PKC-delta might play an important enhancing role in the propagation of phosphotyrosine signals in B cells and mast cells and hence in the regulation of function of both cell types. Therefore, BMMC from PKC-delta -/- mice were generated by in vitro differentiation and assayed for tyrosine phosphorylation events in response to PV. Intriguingly, and opposite to the Rottlerin data, PKC-delta -/- BMMC show a stronger response to PV than wild-type cells, suggesting an attenuating role for PKC-delta. This response can be inhibited equally well by Rottlerin, indicating clearly that Rottlerin is not specific for PKC-delta in vivo. A comparison between Rottlerin and the panspecific PKC inhibitor bisindolylmaleimide suggests that Rottlerin also targets kinases beyond the PKC family. Moreover, Ser473 phosphorylation of protein kinase B (PKB) after PV treatment is blocked by Rottlerin as efficiently as by the phosphatidylinositol 3-kinase inhibitor LY294002. In this report, we provide evidence that PKC-delta constitutes a crucial attenuating factor in B cell and mast cell signal transduction and suggest that PKC-delta is important for the regulation of physiological B and mast cell functions as well as for their pathophysiology. Furthermore, dominant PKC-delta-independent effects of Rottlerin are presented, indicating restrictions of this inhibitor for use in signal transduction research.
Subject(s)
Acetophenones/pharmacology , Benzopyrans/pharmacology , Enzyme Inhibitors/pharmacology , Isoenzymes/physiology , Protein Kinase C/physiology , Vanadates/pharmacology , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Cells, Cultured , Hematopoietic Stem Cells/cytology , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Mast Cells/chemistry , Mast Cells/drug effects , Mast Cells/immunology , Mice , Mice, Knockout , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Phosphotyrosine/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Kinase C-delta , Protein Tyrosine Phosphatases/antagonists & inhibitors , Receptors, IgE/analysis , Tumor Cells, CulturedABSTRACT
Studies in human and rodent models have shown that activation of protein kinase C-beta (PKC-beta) is associated with the development of pathological hypertrophy, suggesting that ablation of the PKC-beta pathway might prevent or reverse cardiac hypertrophy. To explore this, we studied mice with targeted disruption of the PKC-beta gene (knockout, KO). There were no detectable differences in expression or distribution of other PKC isoforms between the KO and control hearts as determined by Western blot analysis. Baseline hemodynamics were measured using a closed-chest preparation and there were no differences in heart rate and arterial or left ventricular pressure. Mice were subjected to two independent hypertrophic stimuli: phenylephrine (Phe) at 20 mg x kg(-1) x day(-1) sq infusion for 3 days, and aortic banding (AoB) for 7 days. KO animals demonstrated an increase in heart weight-to-body weight ratio (Phe, 4.3 +/- 0.6 to 6.1 +/- 0.4; AoB, 4.0 +/- 0.1 to 5.8 +/- 0.7) as well as ventricular upregulation of atrial natriuretic factor mRNA analogous to those seen in control animals. These results demonstrate that PKC-beta expression is not necessary for the development of cardiac hypertrophy nor does its absence attenuate the hypertrophic response.
Subject(s)
Cardiomegaly/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Myocardium/enzymology , Protein Kinase C/genetics , Protein Kinase C/metabolism , Animals , Aorta/physiology , Atrial Natriuretic Factor/genetics , Biomarkers , Blood Pressure/physiology , Carotid Arteries/physiology , Gene Expression Regulation, Enzymologic , Lac Operon , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Kinase C beta , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Ventricular Remodeling/physiology , beta-GalactosidaseABSTRACT
The protein kinase C family of enzymes has been implicated in synaptic plasticity and memory in a wide range of animal species, but to date little information has been available concerning specific roles for individual isoforms of this category of kinases. To investigate the role of the beta isoform of PKC in mammalian learning, we characterized mice deficient in the PKC beta gene using anatomical, biochemical, physiological, and behavioral approaches. In our studies we observed that PKC beta was predominantly expressed in the neocortex, in area CA1 of the hippocampus, and in the basolateral nucleus of the amygdala. Mice deficient in PKC beta showed normal brain anatomy and normal hippocampal synaptic transmission, paired pulse facilitation, and long-term potentiation and normal sensory and motor responses. The PKC beta knock-out animals exhibited a loss of learning, however; they suffered deficits in both cued and contextual fear conditioning. The PKC expression pattern and behavioral phenotype in the PKC beta knock-out animals indicate a critical role for the beta isoform of PKC in learning-related signal transduction mechanisms, potentially in the basolateral nucleus of the amygdala.
Subject(s)
Avoidance Learning/physiology , Brain/metabolism , Conditioning, Psychological/physiology , Fear/physiology , Isoenzymes/genetics , Memory/physiology , Protein Isoforms/genetics , Protein Kinase C/genetics , Amygdala/cytology , Amygdala/metabolism , Animals , Behavior, Animal/physiology , Brain/cytology , Electrophysiology , Hippocampus/cytology , Hippocampus/metabolism , Isoenzymes/metabolism , Long-Term Potentiation/physiology , Mice , Mice, Knockout , Neuronal Plasticity/physiology , Protein Isoforms/metabolism , Protein Kinase C/metabolism , Protein Kinase C beta , Synaptic Transmission/physiologyABSTRACT
Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk(lo)) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk(-/-) mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk(lo) phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk(lo) phenotype in vitro but not in vivo, whereas CD19 and the p85alpha form of phosphoinositide 3-kinase behaved as Btk(lo) enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85alpha haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C beta, Fyn, CD22, Galphaq, or Galpha11 had no detectable effect on the function of Btk(lo) B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.
Subject(s)
B-Lymphocytes/physiology , Protein-Tyrosine Kinases/physiology , Signal Transduction , Tumor Suppressor Proteins , Agammaglobulinaemia Tyrosine Kinase , Animals , Antigens, CD19/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/physiology , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/physiology , Protein Kinase C/physiology , Receptors, Antigen, B-Cell/physiologyABSTRACT
The axial skeleton develops from the sclerotome, a mesenchymal cell mass derived from the ventral halves of the somites, segmentally repeated units located on either side of the neural tube. Cells from the medial part of the sclerotome form the axial perichondral tube, which gives rise to vertebral bodies and intervertebral discs; the lateral regions of the sclerotome will form the vertebral arches and ribs. Mesenchymal sclerotome cells condense and differentiate into chondrocytes to form a cartilaginous pre-skeleton that is later replaced by bone tissue. Uncx4.1 is a paired type homeodomain transcription factor expressed in a dynamic pattern in the somite and sclerotome. Here we show that mice homozygous for a targeted mutation of the Uncx4.1 gene die perinatally and exhibit severe malformations of the axial skeleton. Pedicles, transverse processes and proximal ribs, elements derived from the lateral sclerotome, are lacking along the entire length of the vertebral column. The mesenchymal anlagen for these elements are formed initially, but condensation and chondrogenesis do not occur. Hence, Uncx4.1 is required for the maintenance and differentiation of particular elements of the axial skeleton.
Subject(s)
Axis, Cervical Vertebra/embryology , Homeodomain Proteins/physiology , Ribs/embryology , Animals , Body Patterning , Bone and Bones/abnormalities , Bone and Bones/embryology , Cell Differentiation , Cell Line , Gene Targeting/methods , Homeodomain Proteins/genetics , Mesoderm , Mice , Mice, Knockout , SomitesABSTRACT
Fibrin deposition is a salient feature of hypoxemic vasculature and results from induction of tissue factor. Such tissue factor expression in an oxygen deficient environment is driven by the transcription factor Early Growth Response (Egr)-1. Using homozygous null mice for the protein kinase C beta-isoform gene (PKCbeta null), PKCbeta is shown to be upstream of Egr-1 in this oxygen deprivation-mediated pathway for triggering procoagulant events. Whereas wild-type mice exposed to hypoxia (6%) displayed a robust increase in tissue factor transcripts and antigen, and vascular fibrin deposition, PKCbeta null animals showed a markedly blunted response. Consistent with a central role for Egr-1 in hypoxia-induced expression of tissue factor, PKCbeta null mice subjected to oxygen deprivation displayed at most a minor elevation in Egr-1 transcripts, antigen, and intensity of the gel shift band by electrophoretic mobility shift assay, compared with normoxic animals. These data firmly establish PKCbeta as a trigger for events leading to induction of Egr-1 and tissue factor under hypoxic conditions, and provide insight into a biologic cascade whereby oxygen deprivation recruits targets of PKCbeta and Egr-1, thereby amplifying the cellular response.
Subject(s)
DNA-Binding Proteins/metabolism , Endothelium, Vascular/metabolism , Fibrin/metabolism , Hypoxia/metabolism , Immediate-Early Proteins , Isoenzymes/metabolism , Oxygen Consumption , Protein Kinase C/metabolism , Transcription Factors/metabolism , Animals , Early Growth Response Protein 1 , Enzyme Activation , Gene Expression Regulation, Enzymologic , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Isoenzymes/genetics , Lung/enzymology , Macrophages, Alveolar/enzymology , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/metabolism , Protein Kinase C/genetics , Protein Kinase C betaABSTRACT
We have analysed the involvement of the beta isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-beta is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcgamma-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-beta specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-beta, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-beta also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-beta isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcgamma receptors in neutrophils.
Subject(s)
Isoenzymes/blood , NADPH Oxidases/blood , Neutrophils/physiology , Protein Kinase C/blood , Animals , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Immunoglobulin G , In Vitro Techniques , Indoles/pharmacology , Isoenzymes/antagonists & inhibitors , Kinetics , Latex , Maleimides/pharmacology , Mice , Neutrophils/enzymology , Phagocytosis , Protein Kinase C/antagonists & inhibitors , Protein Kinase C beta , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
The antigen-mediated activation of mast cells by means of IgE antibodies bound to the cell surface leads to direct interactions between FcepsilonRI receptor cytoplasmic domains and various intracellular proteins. These interactions initiate diverse signal-transduction pathways, and the activation of these pathways results in the immediate release of proinflammatory agents. A delayed response also occurs and includes the release of various cytokines. It is clear that the activation of kinases is a requirement for the exocytosis observed in mast cells. In addition to the tyrosine phosphorylation of the affected system by soluble tyrosine kinases, activity of protein kinase C (PKC) results in serine or threonine phosphorylation of multiple protein substrates. In this study, we found that mast cells derived from PKCbeta-deficient mice produce less interleukin 6 in response to IgE-Ag. The inhibition of exocytosis in the PKCbeta-deficient mast cells occurred whether the stimuli were due to the aggregation of the mast cell surface FcepsilonRI or to the calcium ionophore, ionomycin. However, no significant changes were observed in the proliferative response of the mast cells to interleukin 3 (IL-3) or in their apoptotic rate after IL-3 depletion. (Blood. 2000;95:1752-1757)