Calcium-dependent PKC Research Articles

PKC-zeta is essential toward endotoxin-induced macrophage activation

A critical feature of sepsis-induced adult respiratory distress syndrome (ARDS) is the release of cytokines, such as TNF-α, from LPS-activated macrophages. However, the cellular mechanisms involved are incompletely understood. The aim of this study is further clarify the mechanism of LPS activation through the Toll-4 receptor (TLR4) by examining the roles of the various isoforms of PKC within the tissue-fixed macrophage. Methods: RAW 246.7 cells were subjected to LPS stimulation. Selected cells were pretreated with 10 nM Gö6983 to inhibit calcium dependent and novel PKC isoforms, or 60 nM Gö6983 to inhibit atypical PKC isoforms. Following stimulation, cellular and nuclear protein were extracted and analyzed by Western blot and EMS for components of the TLR4 pathway. Supernatants obtained from cells under the various conditions were analyzed by ELISA for the production of TNF-α. Statistical analysis was performed by student t-tests, with a p < 0.05 being significant. Results: LPS stimulation led to the phosphorylation and activation of IRAK, which was followed by activation of JNK, ERK 1/2, and p38. Subsequently, LPS induced the activation of NF-κB and AP-1. Activation of these TLR4-signaling factors was followed by the production of TNF-α. Inhibition of calcium dependent and novel PKC isoforms had no significant effect on any component of TLR4 mediated signaling or TNF-α production. However, inhibition of the atypical PKC, PKC-ζ, was associated with a significant attenuation in the activation of IRAK-1, JNK, ERK 1/2, p38, NF-κB and AP-1 (p<0.05 vs. control for each). This modulation in TLR4 signaling was associated with a significant attenuation of TNF-α (p = 0.001 vs. control). Based on these findings, specific blockade of PKC-ζ was performed using a cell permeable myristoylated PKC-ζ pseudopeptide resulting in similar effects. Conclusion: This study demonstrates that within the macrophage the atypical PKC isoform PKC-ζ is critical to regulation of LPS-induced TLR4-signaling and TNF-α production. Although the mechanism of its activation remains unresolved, it appears that modulation of PKC-ζ activity during gram-negative infections may limit associated inflammatory-induced morbidity, such as ARDS.

Mechanism of enhanced calcium sensitivity and alpha 2-AR vasoreactivity in chronic NOS inhibition hypertension.

PKC augments calcium sensitivity in spontaneously hypertensive rats and contributes to alpha(2)-adrenergic receptor (AR) contraction in rabbit saphenous vein. We showed previously that denuded aortic rings from N(omega)-nitro-l-arginine-treated hypertensive rats (LHR) contract more to CaCl(2) and to the alpha(2)-AR agonist UK-14304 than do rings from normotensive rats (NR). We hypothesized that enhanced PKC activity or a change in PKC isoform contributes to augmented calcium sensitivity and enhanced alpha(2)-AR contraction in LHR aorta. Current studies demonstrate that non-isoform-specific PKC inhibitors reduced UK-14304 contraction in both NR and LHR aorta. However, the calcium-dependent PKC inhibitor Gö-6976 only attenuated contraction in LHR aorta. Additionally, UK-14304 translocated PKC-delta to the membrane in NR aorta, whereas PKC-alpha was translocated to the membrane in LHR aorta. Finally, in ionomycin-permeabilized aorta Gö-6976 eliminated enhanced basal and augmented alpha(2)-AR-stimulated calcium sensitivity in LHR aorta but did not affect NR contraction. Together, these data suggest that PKC-alpha contributes to augmented calcium sensitivity and alpha(2)-AR reactivity after chronic nitric oxide synthase inhibition hypertension.

1,25 (OH) 2D 3 induced rapid increases in calcium dependent PKC activity and intracellular calcium in VDR [-/-] mouse osteoblasts

1,25 (OH) 2D 3 induced rapid increases in calcium dependent PKC activity and intracellular calcium in VDR [-/-] mouse osteoblasts

Concerted regulation of wild-type p53 nuclear accumulation and activation by S100B and calcium-dependent protein kinase C.

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G(1) checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272-4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G(1) phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G(1) arrest at the nonpermissive temperature (37.5 degrees C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32 degrees C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G(1) phase and activation of a p53-dependent G(1) checkpoint control.

Cytosolic hippocampal PKC and aging: correlation with discrimination performance.

Adult and aged mice were submitted to a discrimination task in a radial maze (regular trials), and then to probe trials requiring them to form relational representations. Three weeks later, animals were again tested for regular and probe trials. Following another interval of 3 weeks, individual hippocampal cytosolic calcium-dependent and -independent PKC activities were measured. Performance of aged animals was impaired on probe but not regular trials and aged mice had lower hippocampal cytosolic calcium-dependent and -independent PKC activities than adults. Performance on probe trials was specifically correlated with calcium-dependent PKC activity. This suggests a specific relationship between the ability to form relational representations and hippocampal cytosolic calcium-dependent PKC activity.

Nuclear accumulation of c- myc mRNA in phytohaemagglutinin-activated T lymphocytes treated with anti-HLA class I monoclonal antibody

Anti-HLA class I monoclonal antibody 01.65 inhibits the proliferative response of PHA-activated human T lymphocytes from peripheral blood mononuclear cells. The recruitment rate in the cell cycle is slack and the G1 and S phases are prolonged. Among the early events after PHA activation, only the calcium-dependent PKC activity appears to be modified: particulate PKC is completely depleted while cytosolic residual PKC is reduced by 80% after MAb 01.65 treatment. We have carried out in greater detail the study of c- myc gene regulation by MAb 01.65 and the results are as follows: (i) c- myc RNA transcription is normally initiated and finished, suggesting a post-transcriptional regulation of c- myc gene expression; (ii) no alteration in c- myc mRNA stability has been documented; (iii) steady-state levels of c- myc mRNA expression by Northern blot analysis and PCR amplification are decreased in the cytoplasmic compartment, while in the nuclear compartment they appear to be increased. Nuclear accumulation of mature mRNA after MAb 01.65 and PKC inhibitor (H7 and StSp) treatment appears to be the most probable mechanism involved. The possible implications of this are discussed.

α-Adrenergic Preservation of Myocardial pH during Ischemia Is PKC Isoform Dependent

α-adrenergic stimulation of patients with ischemic heart disease should intuitively impose a destructive stress. However, therapeutic α1-adrenergic receptor mediated cardioadaptation prior to myocardial ischemia protects ventricular mechanical function, promotes electrophysiologic stability, and preserves myocyte viability. Prior to an anticipated cardiac ischemic insult, α1-adrenergic preconditioning attenuates ischemic myocardial acidosis by a protein kinase C-(PKC) dependent mechanism. The α1-adrenoceptor can directly stimulate calcium-independent nPKC isoforms via diacylglycerol (DAG) or indirectly stimulate calcium-dependent cPKC isoforms through the release of intracellular calcium via inositol triphosphate, (IP3). We hypothesized that α1-adrenergic limitation of ischemic acidosis is mediated by the family of calcium-dependent PKC isoforms. [31P]NMR spectra were obtained in isolated, buffer perfused rat hearts treated with α1-adrenergic stimulation [phenylephrine (PE) 50 μM, 2 min]; PKC blockade [chelerythrine chloride, (Chel) 20 μM]; or stearoyl-arachidonoyl glycerol (SAG, a DAG analogue, 100 μM, 2 min) administered 10 min prior to ischemia. Control hearts were perfused under normoxic conditions for 20 min. All hearts were then subjected to global ischemia (20 min, 37.5°C). Developed pressure (DP) and heart rate were recorded continuously. pHiwas obtained from chemical shift of inorganic phosphate. Immunohistochemical staining was utilized to delineate the translocation and activation profiles of specific PKC profiles established with each stimulus. Pre-ischemic α1-adrenergic stimulation did attenuate the myocellular hydrogen ion accumulation during sustained normothermic ischemia (6.90 ± 0.13 vs control 6.54 ± 0.10;P< 0.05). General PKC inhibition abrogated this effect (end-ischemic pH 6.17 ± 0.10;P< 0.05 vs control and PE). Ischemic acidosis was not attenuated following selective nPKC stimulation (SAG, 6.48 ± 0.08; NS vs control). Myocellular immunohistochemical staining revealed translocation of the calcium-independent PKC-ϵ isoform in the calcium-dependent PKC (SAG) group, but not in response to α1-adrenergic stimulation. The results suggest that (1) α1-adrenoceptor stimulation limits ischemic acidosis, (2) α1-adrenergic stimulated attenuation of ischemic acidosis is PKC dependent, (3) direct nPKC stimulation with SAG does not limit ischemic acidosis, and (4) SAG stimulates nPKC-ϵ isoform activation where α1-adrenergic stimulation does not. We conclude that α1-adrenergic stimulation limits ischemic acidosis by a cPKC-dependent mechanism and that the mobilization of the IP3 arm by receptor stimuli suppresses PKC-ϵ thus permitting the limitation of ischemic acidosis.

327 Expression of calcium-dependent and independent PKC isozymes in human basophils (HB)

327 Expression of calcium-dependent and independent PKC isozymes in human basophils (HB)

Anti-CD3-induced changes in protein kinase C isozymes expression in human CD4+ and CD8+ T lymphocytes

In order to determine whether there is a differential expression and activation of PKC isozymes between CD4+ and CD8+ T cells, peripheral blood mononuclear cells were stimulated with anti-CD3 monoclonal antibody (moAb) for various time intervals and the expression of calcium-dependent PKC isozymes (alpha, beta, gamma) and calcium-independent PKC isozymes (delta, epsilon, zeta) was analyzed with dual color flow cytometry, using anti-PKC isozyme antibodies and anti-CD4 or anti-CD8 antibodies. The basal fluorescence intensity of all PKC isozymes was comparable between CD4+ T cells and CD8+ T cells. Following activation with anti-CD3 moAb a marked increase in the fluorescence intensity of all PKC isozymes in both CD4+ and CD8+ T cells, albeit to a different extent and with different kinetics was observed. Among all PKC isozymes studied, the least striking changes were observed in PKC zeta isozyme and the most striking changes were observed in PKC-epsilon isozyme. Laser-based confocal microscopic studies confirmed that the increase in fluorescence intensity of PKC isozymes following anti-CD3 moAb stimulation, as measured by flow cytometry was accompanied by the translocation of PKC isozymes from cytosol to the plasma membrane. This study demonstrates a differential effect of anti-CD3 moAb on the expression of PKC isozymes between CD4+ and CD8+ T cells and suggests that flow cytometry can be used to study the translocation of PKC isozymes from cytosol to the plasma membrane.

Protein kinase C activity, translocation, and conventional isoforms in aging rat brain

Protein kinase C was studied in various brain areas in aging Wistar rats. Histone-directed kinase activity from the cortex, hippocampus and cerebellum did not change with aging. Using purified protein B-50 as a substrate, between 3 and 8 months a decrease in in vitro phosphorylation was detected in the membrane fraction of the cortex but after this age values remained stable. In hippocampal membranes, B-50 phosphorylation was increased in aged rats. PKC translocation was impaired in aged rats in both the cortex and the hippocampus. PKC α and β mRNA decreased in the cortex between 3 and 8 months with no further decline in aged animals. Hippocampal mRNA for calcium-dependent PKC isoforms was not modified during aging, as assessed by Northern and in situ hybridization. Western blot analysis revealed a change in PKC γ protein only, which was increased in hippocampal membranes from aged rats. The data indicate that the key PKC function that is impaired in aged rats is enzyme translocation irrespective of the brain area investigated.