Cytosolic Protein Kinase C Research Articles

Dietary Fat and Fiber Alter Rat Colonic Protein Kinase C Isozyme Expression

To better understand the biochemical mechanisms by which select fats and fibers modulate colonic cell proliferation, we determined the profile of protein kinase C (PKC) isozymes and cell proliferation in rat proximal and distal colonic mucosa following diet manipulation, because enhanced cell proliferation has been correlated with colon cancer incidence. Rats were assigned to one of four diets (each with 15 g fat + 6 g fiber/100 g diet) for 3 wk: fiber-free fish oil (FF), fiber-free corn oil (FC), cellulose + corn oil (CC), or pectin + corn oil (PC). Steady-state levels of colonic mucosal cytosolic and membrane PKC isozymes were determined. In vivo cell proliferation was determined by bromodeoxyuridine incorporation into DNA. In addition, viable exfoliated colonic epithelial cells were isolated from feces using Percoll-bovine serum albumin gradients. We found that 1) proximal and distal colonic mucosa possessed different steady-state levels and relative proportions of PKC isozymes; 2) PKC α and δ expression were significantly greater in distal membrane of the PC-fed group compared with the other dietary groups; 3) the number of exfoliated cells per 4-h fecal collection generally was proportional to the diet-induced changes in cell proliferation (PC>FC>CC>FF). These data demonstrate that dietary treatment altered colonic PKC isozyme expression, with animals fed the fiber-containing diets generally expressing higher steady-state levels of PKC α and δ.

The antitumor natural product tanshinone IIA inhibits protein kinase C and acts synergistically with 17-AAG

Tanshinone IIA (Tan IIA), the primary bioactive compound derived from the traditional Chinese medicine (TCM) Salvia miltiorrhiza Bunge, has been reported to possess antitumor activity. However, its antitumor mechanisms are not fully understood. To resolve the potential antitumor mechanism(s) of Tan IIA, its gene expression profiles from our database was analyzed by connectivity map (CMAP) and the CMAP-based mechanistic predictions were confirmed/validated in further studies. Specifically, Tan IIA inhibited total protein kinase C (PKC) activity and selectively suppressed the expression of cytosolic and plasma membrane PKC isoforms ζ and ε. The Ras/MAPK pathway that is closely regulated by the PKC signaling is also inhibited by Tan IIA. While Tan IIA did not inhibit heat shock protein 90 (Hsp90), it synergistically enhanced the antitumor efficacy of the Hsp90 inhibitors 17-AAG and ganetespib in human breast cancer MCF-7 cells. In addition, Tan IIA significantly inhibited PI3K/Akt/mTOR signaling, and induced both cell cycle arrest and autophagy. Collectively, these studies provide new insights into the molecular mechanisms responsible for antitumor activity of Tan IIA.

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8-37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8-37-cholestanol, but not unconjugated CGRP8-37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8-37-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention.

Effect of α-linolenic acid-rich diacylglycerol oil on protein kinase C activation in the rat digestive tract and lingual mucosa

1,2-Diacylglycerol with short chain fatty acids is an endogenous activator of protein kinase C (PKC), which involved in multiple cellular processes implicated in cancer. The aim of the present study was to assess the effects of dietary α-linolenic acid-rich diacylglycerol (ALA-DAG) oil on PKC activation in the rat digestive tract and lingual mucosa in comparison with the effects of α-linolenic acid-rich triacylglycerol (ALA-TAG) oil, and common dietary oil. Membranous PKC activity in the lingual mucosa of male Wistar rats was significantly activated by treatment of the tongue with 1,2-tetradecarnoylphorbol-13-acetate (100 μM) twice in 1 day. In contrast, animals consuming a diet containing either ALA-DAG oil (7.5% or 30%), ALA-TAG oil (7.5% or 30%), or rapeseed oil (30%) for 4 weeks exhibited no significant differences in the cytosolic and membrane PKC activity in the lingual, esophageal, gastric, small intestinal, and colonic mucosa. Dose-related increases in PKC activity were not observed in the ALA-DAG oil-fed groups. Thus, the effects of dietary ALA-DAG oil on PKC activation in the digestive tract and lingual mucosa was similar to those of the ALA-TAG and rapeseed oils. These findings suggest that replacement of common dietary oil with ALA-DAG oil would not increase the risk of carcinogenesis.

Arachidonic acid-induced down-regulation of protein kinase C delta in beta-cells.

We have previously identified expression of multiple protein kinase C (PKC) isoforms in insulinoma-derived beta-cells and whole islets. Both PKC delta and PKC alpha appear to be the more abundantly expressed isoforms. In this report we studied the effects of arachidonic acid (AA) on the subcellular distribution of PKC alpha and PKC delta. AA has been reported to activate both PKC alpha and PKC delta and it is thought to be an important second messenger in beta-cells. Here we report that AA interacted with and altered beta-cell pools of PKC delta preferentially over PKC alpha. AA (100 microM) over the course of 45 min reduced cytosolic levels of PKC delta (to 40 +/- 15%, compared to time zero control) leaving membrane- and cytoskeleton-associated levels near control levels. Analysis of whole cell homogenates showed a slight down-regulation of PKC delta indicating proteolysis. The down-regulation of cytosolic PKC delta appeared to be isoform specific since cytosolic PKC alpha remained at control levels over the time course. The response was dose-dependent and negligible at concentrations below 30 microM and occurred, at least partially, in the cytosolic compartment of the cell. Indomethacin also down-regulated cytosolic PKC delta preferentially over PKC alpha possibly through accumulation of AA. These findings suggest that cytosolic PKC delta may be a downstream target of this beta-cell second messenger.

C‐kit mutations and PKC crosstalks: PKC translocates to nucleous only in cells HMC560,816

The human mast cell lines HMC-1(560) and HMC-1(560,816) were used to study histamine release, Ca(2+) signaling and protein kinase C (PKC) localization and expression, with phorbol 12-myristate 13-acetate (PMA). Both sublines carry activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit tyrosine kinase activation. Both have the Gly-560 → Val mutation but only the second carries the Asp-816 → Val mutation. In this study, it was observed that the stimulation of PKC has different effects in HMC-1(560) and HMC-1(560,816) and this would be related to the difference in activating mutations in both mast cell lines. PKC activation increases ionomycin-induced histamine release in HMC-1(560) . This article demonstrates an opposite histamine response in HMC-1(560,816) cells, even though classical PKCs are the family of isozymes responsible for this effect in both cellular lines. Furthermore, it can be observed that upon cell stimulation with PMA, primarily cytosolic PKC translocates to the nucleous in HMC-1(560,816) cells, but not in HMC-1(560) cell line.

Phorbol dibutyrate induces contractions in bovine cerebral arteries by an extracellular calcium-independent mechanism.

The aim of the present study was to analyse the ability of phorbol 12,13-dibutyrate (PDB) to activate protein kinase C (PKC), measured by its capacity to translocate the enzyme from the cytosol to the membrane fraction, as well as to induce vasconstrictive responses in segments from branches of bovine cerebral arteries. PDB (0.1 microM) produced a marked translocation of PKC activity from the cytosolic to the membranous fraction. This drug induced concentration-dependent contractions which were slow in onset. The contraction elicited by PDB was reduced by the PKC inhibitor, staurosporine (1 and 10 nM), but unaltered by both Ca(2+)-free medium containing 3 mM EGTA and the Ca(2+)-channel antagonist, nifedipine (1 microM). Preincubation of segments with PDB (10 and 30 nM) reduced the vasoconstriction elicited by 5-hydroxytryptamine (5-HT) in a concentration- and preincubation time-dependent manner. These data indicate that bovine cerebral arteries possess cytosolic and membranous PKC activities, that the vasoconstrictive responses induced by PDB were independent of extracellular Ca2+, that cytosolic C-kinase is translocated to the membrane and probably down-regulated by PDB, and that this enzyme is not involved in 5-HT responses, but is down-regulated by PDB.

Possible involvement of protein kinase c inhibition in the reduction of phorbol ester-induced neutrophil aggregation by magnolol in the rat.

The influence of the plant product magnolol on neutrophil aggregation has been investigated in the rat. Magnolol inhibited phorbol 12-myristate 13-acetate (PMA)-activated rat neutrophil aggregation in a concentration-dependent manner with an IC50 (concentration resulting in 50% inhibition) of 24.2 +/- 1.7 microM. Magnolol suppressed the enzyme activity of neutrophil cytosolic and rat brain protein kinase C (PKC) over the same range of concentrations at which it inhibited the aggregation. Magnolol did not affect PMA-induced cytosolic PKC-alpha and -delta membrane translocation or trypsin-treated rat-brain PKC activity, but attenuated [3H]phorbol 12,13-dibutyrate binding to neutrophil cytosolic PKC. These results suggest that the inhibition of PMA-induced rat neutrophil aggregation by magnolol is probably attributable, at least in part, to the direct suppression of PKC activity through blockade of the regulatory region of PKC.

Regulation of Prostate Cancer Cell Survival by Protein Kinase C∈ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.

Neurobehavioral teratogenicity of perfluorinated alkyls in an avian model

Perfluorinated alkyls are widely-used agents that accumulate in ecosystems and organisms because of their slow rate of degradation. There is increasing concern that these agents may be developmental neurotoxicants and the present study was designed to develop an avian model for the neurobehavioral teratogenicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Fertilized chicken eggs were injected with 5 or 10mg/kg of either compound on incubation day 0. On the day of hatching, imprinting behavior was impaired by both compounds. We then explored underlying mechanisms involving the targeting of protein kinase C (PKC) isoforms (α, β, γ) in the intermedial part of the hyperstriatum ventrale, the region most closely associated with imprinting. With PFOA exposure, cytosolic PKC concentrations were significantly elevated for all three isoforms; despite the overall increase in PKC expression, membrane-associated PKC was unaffected, indicating a defect in PKC translocation. In contrast, PFOS exposure evoked a significant decrease in cytosolic PKC, primarily for the β and γ isoforms, but again without a corresponding change in membrane-associated enzyme; this likely partial, compensatory increases in translocation to offset the net PKC deficiency. Our studies indicate that perfluorinated alkyls are indeed developmental neurotoxicants that affect posthatch cognitive performance but that the underlying synaptic mechanisms may differ substantially among the various members of this class of compounds, setting the stage for disparate outcomes later in life.

Oxalate-induced activation of PKC-α and -δ regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC-alpha and -delta from the cytosol to the cell membrane. Pretreatment of LLC-PK1 cells with specific inhibitors of PKC-alpha or -delta significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK1 cells as well as cytosol-to-membrane translocation of PKC-alpha and -delta. Silencing of PKC-alpha expression by PKC-alpha-specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-alpha- and -delta-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers.

Activation of human platelet protein kinase C-beta 2 in vivo in response to acute hyperglycemia.

Protein kinase C (PKC) is known to be activated in experimental model systems by elevated glucose and may play an important role in the pathogenesis of diabetic complications. Since there is no information about its role in humans in vivo we investigated the activation of PKC in human thrombocytes during infusion of glucose and insulin in normal controls and in 19 NIDDM patients by determining membrane and cytosol levels of PKC beta 2 using immune blots. In the 27 subjects investigated (8 controls, 19 NIDDM) membrane-associated levels of PKC beta 2 increased significantly after 60 and 150 min (p < 0.005). In controls an increase of membrane and of cytosolic PKC beta 2 occurred upon elevation of glucose by 5.5 mmol/L or more and the membrane association persisted for at least 60 min. In NIDDM glucose was elevated by 7.5-10 mmol/L during infusions. Increases of both membrane and cytosolic PKC beta 2 (< 20%-300%) occurred in 10 NIDDM patients suggesting that both, translocation and increased synthesis of PKC beta 2 were stimulated by glucose. Nine other patients showed no alteration (i.e. < 20%) of PKC beta 2. The 2 groups were similar regarding parameters of diabetes control, baseline glucose and glucose elevation during the test. However, the PKC beta 2 responsive group had lower levels of serum triglycerides (1.39 +/- 0.19 vs. 2.32 +/- 0.34 g/L; p = 0.038). To assess whether absolute levels of PKC were altered in human diabetes, platelet levels of PKC alpha, beta 1 and beta 2 were determined in 22 controls and 25 NIDDM subjects with poorly controlled diabetes (HbA1c = 9.8 +/- 0.36%). Cytosolic levels of PKC alpha were significantly decreased by 27% compared to controls in NIDDM but there was no change of PKC beta 1 or PKC beta 2. We conclude that 1. acute elevation of glucose by 5.5 mmol/L or more can activate PKC beta 2 translocation in controls and NIDDM patients in vivo irrespective of parameters of metabolic control. 2. NIDDM patients differ in their PKC beta 2-responses to glucose and 3. poor metabolic control leads to moderate downregulation of PKC alpha suggesting continued activation.

Protein Kinase C

Protein kinase C (PKC) is involved in tumor growth and apoptosis and hence represents a potential target for cancer therapy. This study investigated the expression of PKC in pancreatic tumor tissue in comparison to adjacent normal tissue and determined the modulation of PKC by bryostatin-1 (BRYO) on pancreatic cancer cell lines. Pancreatic tissue was obtained from 18 patients who had a resection (14 with ductal adenocarcinoma and 4 with adenoma and high-grade dysplasia). Cytosolic and nuclear membrane PKCs in the paired samples were determined by immunoblotting. HPAC cells were treated with gemcitabine and BRYO and in sequential and concomitant combination. To evaluate cell viability, apoptosis, and electrophoretic mobility shift assay, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, enzyme-linked immunosorbent assay, and nuclear factor kappaB (NF-kappaB) assays were used. As compared with the adjacent normal tissue, PKC-alpha, PKC-beta1, and PKC-delta were higher in the tumor; PKC-epsilon was higher in the normal tissue. Pretreatment with gemcitabine followed by BRYO resulted in decreased cell viability, increased apoptosis, and inhibited NF-kappaB than either agent alone or BRYO followed by gemcitabine. Protein kinase C is overexpressed and activated in pancreatic cancer as compared with normal tissue. Inhibition of PKC could sensitize pancreatic cancer cell lines to the effects of gemcitabine. The potentiation of gemcitabine by BRYO is sequence-dependent and mediated through inhibition of PKC-dependent activation of NF-kappaB.

Protein Kinase C Activity and Cell Proliferation in Polycystic Kidney Disease

Polycystic kidney disease is associated with the loss of the structure/function of the primary cilia. Cilia regulate intracellular calcium concentration ([Ca2+]i), with the loss of cilia leading to an elevation in [Ca2+]i. These studies examine whether this increase in [Ca2+]i would lead to protein kinase C (PKC) activation and whether activated PKC would contribute to cell proliferation in polycystic kidney disease. Studies were performed in cilia (‐) and cilia (+) collecting duct cell lines, derived from the Tg737orpk mouse. Cilia (‐) cells compared to cilia (+) cells displayed reduced cytosolic PKC content. There was also increased translocation of PKC to the plasma membrane in cilia (‐) cells, as assessed with a α‐PKC antibody and confocal microscopy. In other studies, cilia (‐) cells proliferated at a much higher rate than cilia (+) cells. Chronic treatment with 10 μM BAPTA‐AM, a Ca2+ chelating agent, reduced the proliferation rate in cilia (‐) cells to a level comparable to cilia (+) cells. Importantly, inhibition of α‐PKC with Ro‐32‐0432, also abolished increased proliferation in cilia (‐) cells compared to cilia (+) cells. Thus, we suggest that there is Ca2+‐dependent increased α‐PKC translocation in cilia (‐) cells and that elevated PKC activity contributes to the hyper‐proliferation and possibly cyst formation in this model of polycystic kidney disease.

Inhibition of NADPH Oxidase Prevents Advanced Glycation End Product–Mediated Damage in Diabetic Nephropathy Through a Protein Kinase C-α–Dependent Pathway

Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in protein kinase C (PKC) and increased advanced glycation was examined. Streptozotocin-induced diabetic or nondiabetic Sprague Dawley rats were followed for 32 weeks, with groups randomized to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg . kg(-1) . day(-1); weeks 16-32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of advanced glycation end products (AGEs)-BSA, were treated with either apocynin or the PKC-alpha inhibitor Ro-32-0432. RESULTS; Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic, and skin collagen-associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-alpha to renal membranes, was associated with increased NADPH-dependent superoxide production and elevated renal, serum, and urinary vascular endothelial growth factor (VEGF) concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-alpha attenuated cytosolic superoxide and PKC activation and increased VEGF. Finally, renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin. In the context of these and previous findings by our group, we conclude that activation of NADPH oxidase via phosphorylation of PKC-alpha is downstream of the AGE-receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.

Apple Polyphenols Affect Protein Kinase C Activity and the Onset of Apoptosis in Human Colon Carcinoma Cells

Polyphenol-rich apple extracts have been reported to suppress human colon cancer cell growth in vitro. The protein kinase C (PKC) is among the signaling elements known to play an important role in colon carcinogenesis. In the present study, we investigated whether apple polyphenols affect PKC activity and induce apoptosis in the human colon carcinoma cell line HT29. A polyphenol-rich apple juice extract (AE02) was shown to inhibit cytosolic PKC activity in a cell-free system. In contrast, incubation of HT29 cells for 1 or 3 h with AE02 up to 2 mg/mL did not affect the cytosolic PKC activity. After prolonged incubation (24 h), cytosolic PKC activity was modulated, albeit a u-shaped curve of effectiveness was observed, with an initial inhibitory effect followed by the recurrence and even induction of enzyme activity. Concomitantly, in the cytosol, a significant decrease of the protein levels of PKCalpha, PKCbetaII, and PKCgamma together with a significant increase of a proapoptotic PKCdelta fragment was observed. However, the effects on the protein levels of these PKC isoforms in the cytosol were not associated with translocation between the different cellular compartments but might instead result from the onset of apoptosis. Indeed, the treatment with AE02 was shown to induce apoptosis by the activation of caspase-3, DNA fragmentation, and cleavage of poly(ADP ribose) polymerase. So far, identified and available constituents of the apple extract did not contribute substantially to the observed effects on PKC and apoptosis induction. In summary, apple polyphenols were found to inhibit PKC activity in a cell-free system. However, our results indicate that within intact cells PKC does not represent the primary target of apple polyphenols but appears to be affected in the course of apoptosis induction.

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Overexpression of amyloid precursor protein reduces epsilon protein kinase C levels

Alzheimer’s disease (AD) is characterized by extracellular deposits of amyloid beta peptide (Aβ), a peptide that is generated upon proteolytic cleavage of amyloid precursor protein (APP). The events leading to the development of AD and their sequence are not yet fully understood. Protein kinase C (PKC) has been suggested to have a significant role in controlling neuronal degeneration and in the aberrant signal transduction taking place in AD. Several studies document a deficit in PKC levels and activity in brains of AD patients when compared with those of normal controls. Such a decrease in PKC could have serious implications since certain PKC isozymes were shown to drive the APP proteolytic cleavage into a non-amyloidogenic pathway. Reduced levels of distinct PKC isozymes could thus contribute to driving APP processing toward an amyloidogenic pathway. The direct cause for the down-regulation of PKC in AD brains is still unknown. In that respect, we tested in this study whether APP may play a role in PKC reduction. We show in three different cell lines (CHO, COS and BOSC) that overexpression of APP leads to decreased PKC levels. This decrease was found to be specific for the epsilon PKC isozyme whereas the levels of delta, alpha and conventional PKC remained unchanged. Furthermore, we observed this decrease for both active, membrane-associated and inactive, cytosolic epsilon PKC. APP-driven decrease in epsilon PKC is most likely mediated by a factor in the culture medium, since transfer of medium from cultured cells overexpressing APP to naïve, non-overexpressing cells, has also led to the selective decrease in epsilon PKC levels. Taken together, our results suggest that APP expression levels may play a role in the decrease of epsilon PKC levels in AD brains and could thus affect the responsiveness of AD brain tissues to growth factors and neurotransmitters.

Comparison of dietary triacylglycerol oil and diacylglycerol oil in protein kinase C activation

The objective of the present study was to compare the effects of dietary diacylglycerol (DAG) oil with triacylglycerol (TAG) oil with a similar fatty acid composition (fatty acid chain range: C14–C22, C18 fatty acid chain: >90%) on protein kinase C (PKC) activation and on 1,2-DAG levels. Using male Wistar rats, no differences in cytosolic and membrane PKC activities in the lingual, esophageal, gastric, small intestinal, cecal, proximal colonic, and distal colonic mucosa were found between the 5% DAG and TAG oil groups, or between the 23% DAG and TAG oil groups after 1 month of feeding. The 1,2-DAG levels in the cecum and colon contents and in the feces and serum in male Wistar rats after a diet containing either 10% DAG or TAG oil feeding were similar between the groups. Moreover, exposure of Caco-2 cells to DAG and TAG oils had no effect on PKC activity in the membrane fraction, but 1,2-dioctanoyl glycerol composed of short-chain fatty acids (C8) did, suggesting the absence of an influence on PKC activity in DAG and TAG oils composed of long-chain fatty acids. In summary, the effects of DAG oil ingestion on PKC activity in the digestive tract and lingual mucosa, and on 1,2-DAG levels in the cecum and colon contents and in the feces and serum were similar to those observed for TAG oil ingestion.

Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Cα in C2C12 myocytes

Protein kinase C (PKC) agonists including phorbol 12-myristate 13-acetate (PMA) not only induce the redistribution of cytosolic PKC to various subcellular compartments but also activate the kinase domain of the protein. In the present study we have investigated the nature of mitochondrial PKC pool and its effects on mitochondrial function in cells treated with PMA. Treatment of C2C12 myoblasts, C6 glioma and COS7 cells with PMA resulted in a dramatic redistribution of intracellular PKCα pool, with large fraction of the protein pool sequestered in the mitochondrial compartment. We also observed mitochondrial PKCδ accumulation in a cell restricted manner. The intramitochondrial localization was ascertained by using a combination of protection against protease treatment of isolated mitochondria and immunofluorescence microscopy. PMA-induced mitochondrial localization of PKCα was accompanied by increased mitochondrial PKC activity, altered cell morphology, disruption of mitochondrial membrane potential, decreased complex I and pyruvate dehydrogenase activities, and increased mitochondrial ROS production. All of these changes could be retarded by treatment with PKC inhibitors. These results show a direct role for PMA-mediated PKCα translocation to mitochondria in inducing mitochondrial toxicity.