Protein Kinase C Enzyme Activity Research Articles

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.

Expression of protein kinase C in relation to the embryonic diapause process in the silkworm, Bombyx mori

In the present study, we investigated the expression of protein kinase C (PKC) signaling during the embryonic diapause process of Bombyx mori. PKC activity, determined using an antibody to phosphorylated substrates of PKC, was found to be significantly higher in developing eggs as compared to that of diapause eggs. In eggs whose diapause initiation was prevented by HCl, non-diapause eggs, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C, PKC-dependent phosphorylation levels of multiple proteins showed dramatic stage-dependent increases compared to those of diapause eggs. Higher protein levels of PKC were also detected in developing eggs as compared to those of diapause eggs. Determination of PKC enzymatic activity during the middle embryonic stage showed higher PKC activity in developing eggs compared to diapause eggs, thus further confirming differential regulation of PKC signaling during the embryonic diapause process. Examination of temporal changes in mRNA expression levels of classical PKC (cPKC) and atypical PKC (aPKC) showed no difference between diapause and HCl-treated eggs. These results demonstrated that differential expressions of PKC signaling between diapause and developing eggs are related to the embryonic diapause process of B. mori.

Ca2+ Requirements for Long-Term Depression Are Frequency Sensitive in Purkinje Cells.

Cerebellar long-term depression (LTD) is a form of synaptic plasticity dependent on postsynaptic Ca2+ changes. One fundamental question is how LTD is selectively induced by specific numbers of Ca2+ pulses and which are the frequency and duration of this train of pulses required for LTD induction. The molecular mechanism which leads the integration of postsynaptic Ca2+ pulses in the LTD signaling network has not been elucidated either. Recent publications have shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is required for LTD induction. Additionally, protein kinase C (PKC), CaMKII, and MAPK play an important role to transduce the frequency of Ca2+ pulses into their enzymatic activity levels; however, it is still unknown which enzymes are involved in decoding Ca2+ pulses in LTD. We have extended a stochastic model of LTD by adding the molecular network regulating CaMKII activity and its activation. We solved this model with stochastic engine for pathway simulation to include the effect of biochemical noise in LTD. We systematically investigated the dependence of LTD induction on stimulus frequencies, and we found that LTD is selectively induced by a specific number of Ca2+ spikes at different frequencies. We observed that CaMKII is essential to induce LTD, and LTD is only weakly induced when its Thr286 phosphorylation site has been deleted. We found that CaMKII decodes the frequency of Ca2+ spikes into different amounts of kinase activity during LTD induction. In addition, PKC and ERK enzyme activity is highly sensitive to the frequency and the number of Ca2+ pulses and this sensitivity has an important effect on LTD activation. This research predicts the postsynaptic Ca2+ requirements to induce LTD using a typical synaptic activation sequence and explains how LTD is selectively induced by specific number of Ca2+ pulses at different frequencies.

PKC activation sensitizes basal-like breast cancer cell lines to Smac mimetics

There is a need for novel strategies to initiate cancer cell death. One approach is the use of Smac mimetics, which antagonize inhibitor of apoptosis proteins (IAPs). Recent studies have shown that combinations of Smac mimetics such as LBW242 or LCL161 in combination with chemotherapeutic agents increase cancer cell death. Here we show that the protein kinase C (PKC) activator TPA together with the Smac mimetic LBW242 induces cell death in two basal breast cancer cell lines (MDA-MB-468 and BT-549) that are resistant to Smac mimetic as single agent. Ten other LBW242-insensitive cancer cell lines were not influenced by the TPA+LBW242 combination. The TPA+LBW242 effect was suppressed by the PKC inhibitor GF109203X, indicating dependence on PKC enzymatic activity. The PKC effect was mediated via increased synthesis and release of TNFα, which can induce death in the presence of Smac mimetics. The cell death, coinciding with caspase-3 cleavage, was suppressed by caspase inhibition and preceded by the association of RIP1 with caspase-8, as seen in complex II formation. Smac mimetics, but not TPA, induced the non-canonical NF-κB pathway in both MDA-MB-231 and MDA-MB-468 cells. Blocking the canonical NF-κB pathway suppressed TPA induction of TNFα in MDA-MB-468 cells whereas isolated downregulation of either the canonical or non-canonical pathways did not abolish the Smac mimetic induction of the NF-κB driven genes TNFα and BIRC3 in MDA-MB-231 cells although the absolute levels were suppressed. A combined downregulation of the canonical and non-canonical pathways further suppressed TNFα levels and inhibited Smac mimetic-mediated cell death. Our data suggest that in certain basal breast cancer cell lines co-treatment of TPA with a Smac mimetic induces cell death highlighting the potential of using these pathways as molecular targets for basal-like breast cancers.

Amyloid beta peptide (1-42)-mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells.

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.

Abstract 2285: PKC activation sensitizes certain basal breast cancer cell lines to the Smac mimetic LBW242

Abstract There is a need for novel strategies to initiate cancer cell death. One approach is the use of Smac mimetics, such as LBW242, which antagonize inhibitor of apoptosis proteins (IAP)s and facilitates cell death. Recent studies have shown that combinations of Smac mimetics and chemotherapeutic agents increase cancer cell death. Here we show that the protein kinase C (PKC) activator TPA together with LBW242 induce cell death in two basal breast cancer cell lines (MDA-MB-468 and BT549), that are resistant to LBW242 as a single agent. Eleven other LBW242-insensitive cancer cell lines were not influenced by the TPA+LBW242 combination. The TPA+LBW242 effect was suppressed by the PKC inhibitor GF109203X, indicating dependence on PKC enzymatic activity. The PKC effect was mediated via increased synthesis and release of TNFα which can induce death in the presence of LBW242. The cell death, coinciding with caspsae-3 cleavage, was suppressed by caspase inhibition and preceded by the association of RIP1 with Caspase-8, as seen in complex II mediated apoptosis. Our data suggest that in certain basal breast cancer cell lines co-treatment of TPA with LBW242 induces cell death highlighting the potential of using these pathways as molecular targets for basal-like breast cancers. Citation Format: Louise Cornmark, Christian Holmgren, Katarzyna Masoumi, Christer Larsson. PKC activation sensitizes certain basal breast cancer cell lines to the Smac mimetic LBW242. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2285. doi:10.1158/1538-7445.AM2014-2285

Protein kinase C-dependent signaling controls the midgut epithelial barrier to malaria parasite infection in anopheline mosquitoes.

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.

Increased anxiety-related behaviour in Hint1 knockout mice

Several reports have implicated a role for the histidine triad nucleotide-binding protein-1 (Hint1) in psychiatric disorders. We have studied the emotional behaviour of male Hint1 knockout (Hint1 KO) mice in a battery of tests and performed biochemical analyses on brain tissue. The behavioural analysis revealed that Hint1 KO mice exhibit an increased emotionality phenotype compared to wildtype (WT) mice, while no significant differences in locomotion or general exploratory activity were noted. In the elevated plus-maze (EPM) test, the Hint1 KO animals entered the open arms of the apparatus less often than WT littermates. Similarly, in the dark–light box test, Hint1 KO mice spent less time in the lit compartment and the number of entries were reduced, which further confirmed an increased anxiety-related behaviour. Moreover, the Hint1 KO animals showed significantly more struggling and less floating behaviour in the forced swim test (FST), indicating an increased emotional arousal in aversive situations. Hint1 is known as a protein kinase C (PKC) interacting protein. Western blot analysis showed that PKCγ expression was elevated in Hint1 KO compared to WT mice. Interestingly, PKCγ mRNA levels of the two groups did not show a significant difference, implying a post-transcriptional PKCγ regulation. In addition, PKC enzymatic activity was increased in Hint1 KO compared to WT mice. In summary, our results indicate a role for Hint1 and PKCγ in modulating anxiety-related and stress-coping behaviour in mice.

Invasion ofCryptococcus neoformansinto human brain microvascular endothelial cells requires protein kinase C-α activation

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCalpha in HBMEC. During C. neoformans infection, phosphorylation of PKCalpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCalpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCalpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCalpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCalpha and downstream actin filament activity during the fungal invasion into HBMEC.

Differential signalling pathways involved in cholinoceptor‐dependent stimulation of nitric oxide isoforms in dental pulp

To investigate the role of muscarinic acetylcholine receptor (mAChR) subtype activity in the regulation of endothelial- (e) and neuronal- (n) nitric oxide synthase (NOS) expression and activity. Rat dental pulp tissue was used throughout the study. The e-nos and n-nos mRNA levels were specifically measured using reverse transcriptase polymerase chain reaction procedures that involve simultaneous co-amplification of both target cDNA and a reference template with a single set of primers. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. Stimulation of M(1)/M(2) and M(3)/M(4) mAChRs with pilocarpine caused an increase in e-nos and n-nos mRNA levels and NOS activity in the dental pulp. The specific mAChR subtype antagonists, L-NMMA, l-NIO and N(2)-propyl-L-arginine but not aminoguanidine attenuated all these effects. Inhibitors of phospholipase C (PLC), protein kinase C (PKC) and calcium/calmodulin (CaM) prevented the pilocarpine-dependent increase in n-nos and e-nos mRNA levels and NOS activity. Activation of mAChR subtypes stimulated NOS activity by increasing the production of NO through e-nos and n-nos gene expression and NOS activity. The mechanism appears to occur secondarily to stimulation of CaM and PKC enzymatic activity.

Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation

PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131–139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant α, β 1, β 2, γ, δ, ζ and η PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of α, β 1 and β 2 PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.

Human Neutrophil Elastase Induces Hypersecretion of Mucin from Well-Differentiated Human Bronchial Epithelial Cells in Vitro via a Protein Kinase Cδ-Mediated Mechanism

The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC 5 AC and MUC 5 B, but not MUC 2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCalpha, delta, zeta, lambda, iota, and epsilon were constitutively expressed in NHBE cells while PKCbeta, eta, and mu were PMA-inducible. PKCdelta was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCdelta attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the delta isoform.

Inducible nitric oxide synthase subserves cholinergic vasodilation in retina

In this paper, we investigate the role of muscarinic acetylcholine receptor (mAChR) activity in the regulation of inducible (i) nitric oxide synthase (iNOS) expression and activity. The signaling pathway involved is also examined. These experiments also provide a link between mAChR activation and the nitric oxide (NO)-dependent regulation of retinal vascular diameter. The diameter of the retinal vessels at a distance of 1 disc diameter from the center of the optic disc was measured in rats using digital retinal photography, and both iNOS-mRNA gene expression and NOS were specifically measured using RT-PCR and [U-(14)C] citrulline assays, respectively. Stimulation of M(1) and M(3) mAChR with carbachol caused an increase in vessel diameter, in iNOS-mRNA levels and in NOS activity in the retina. Aminoguanidine, an inhibitor of iNOS, attenuated all these effects. Inhibitors of phospholipase C (PLC) and protein kinase C (PKC) but not calcium/calmodulin (CaM) prevented the muscarinic-dependent increase in iNOS-mRNA levels. The results obtained suggest that the activation of mAChR increases retinal vessel diameters by increasing the production of nitric oxide (NO) through iNOS activation and iNOS-mRNA gene expression. The mechanism appears to occur secondarily to stimulation of PLC and PKC enzymatic activity.

The golli-myelin basic protein negatively regulates signal transduction in T lymphocytes

Protein kinase C (PKC) plays a critical role in signal transduction controlling T lymphocyte activation. Both positive and negative regulation of signal transduction is needed for proper control of T lymphocyte activation. We have found that a golli product of the myelin basic protein (MBP) gene can serve as a negative regulator of signaling pathways in the T lymphocyte, particularly the PKC pathway. Increased expression of golli BG21 in Jurkat T cells strongly inhibits anti-CD3ε-induced IL-2-luciferase activity, an indicator of T lymphocyte activation. Golli BG21 can be phosphorylated by PKC in vitro and its phosphorylation increases in PMA-activated Jurkat cells. BG21 inhibits the PMA-induced increase in AP-1 or NF-κB activation, consistent with golli acting in a PKC-mediated cellular event. Golli BG21 inhibition of the PKC pathway is not due to a direct action on PKC activation but in the cascade following PKC activation, since BG21 neither reduces PKC enzyme activity nor blocks the membrane association of PKCθ brought on by T lymphocyte activation. The inhibitory function of BG21 is independent of its phosphorylation by PKC because a mutant BG21, in which the PKC sites have been mutated, is as effective as the wild type BG21 in inhibiting the PMA-induced AP-1 activation. Structure–function assays indicate that BG21 inhibitory activity resides in the golli domain rather than in MBP domain of the molecule. These results reveal a novel role for MBP gene products in T lymphocytes within the immune system.

Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-κB and tumor necrosis factor α in LLC-PK1 cells

Fumonisin B1 (FB1), the most potent of the fumonisin mycotoxins, is a carcinogen and causes a wide range of species-specific toxicoses. FB1 modulates the activity of protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases that play important role in modulating a variety of biologic responses ranging from regulation of cell growth to cell death. Although it has been demonstrated that FB1 induces apoptosis in many cell lines, the precise mechanism of apoptosis is not fully understood. In this study, we investigated the membrane localization of various PKC isoforms, PKC enzyme activity, and its downstream targets, namely nuclear factor-kappa B (NF-κB), tumor necrosis factor α (TNFα), and caspase 3, in porcine renal epithelial (LLC-PK1) cells. FB1 repressed cytosol to membrane translocation of PKC-α, -δ, -ε, and -ζ isoforms over 24–72 h. The FB1-induced membrane PKC repression was corroborated by a concentration-dependent decrease in total PKC activity. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) for this duration also resulted in repressed PKC membrane localization and activity comparable to FB1. Exposure of cells to FB1 (10 μM) was associated with inhibition of cytosol to nuclear translocation of NF-κB and NF-κB-DNA binding at 72 h. The expression of TNFα was significantly inhibited at 24 and 48 h in response to 1 and 10 μM FB1. Increased caspase 3 activity was observed in LLC-PK1 cells exposed to ≥1 μM FB1 at 48 h. PMA also increased the caspase 3 activity at 24 and 48 h. Results suggest that FB1-induced apoptosis involves the activation of caspase 3, which is associated with the repression of PKC and possibly its down-stream effectors, NF-κB and TNFα.

Vitamin A (Retinoids) Regulation of Mouse Melanoma Growth and Differentiation

The incidence of melanoma is rapidly increasing in the U.S. population. At the present, there is no effective chemotherapy against invasive melanoma. At our laboratory, we have been studying retinoic acid (RA)-induced growth arrest and differentiation in the B16 murine melanoma cell model. Several immediate-early gene targets of RA were identified by gene arrays. In one of these genes, T-box binding protein-2 (Tbx-2), an RA response element, was identified in the promoter region that mediates the RA responsiveness of this gene. RA also induces a sixfold to eightfold increase in protein kinase C (PKC)alpha RNA and protein. This gene is not a direct target of RA but appears to be required for the biological effects of RA in B16 melanoma cells. PKC can alter gene transcription via phosphorylation of activator protein (AP)-1. RA increased AP-1 activity in B16 cells but with delayed kinetics compared with activation of PKC by phorbol dibutyrate. Clones stably expressing a dominant negative A-fos gene had reduced AP-1 activity and were less sensitive to RA induction of growth arrest and differentiation. Paradoxically, although inhibition of PKC enzyme activity blocked phorbol dibutyrate-stimulated AP-1 activity, it had no effect on RA-induced AP-1 activity. Further investigation showed that PKC enzyme activity was not required for RA-induced growth inhibition or stimulation of melanin synthesis. These data suggest that PKCalpha either works through a nonenzymatic protein-protein mechanism or may interfere with the enzymatic function of another isozyme of PKC to mediate the actions of RA in B16 melanoma cells.

Differential regulation of cardiac protein kinase C isozyme expression after aortic banding in rat.

Protein kinase C (PKC) plays a key role in myocardial hypertrophy. To evaluate whether its isoforms are expressed differentially during gradual development of pressure-overload-induced cardiac hypertrophy, banding of the ascending aorta was used as an experimental model of left ventricular hypertrophy. One, 7 and 30 days after sham operation or aortic banding in male Wistar rats, the PKC activity and the expression of the cardiac PKC isozymes (PKC-alpha, -delta, - epsilon and -zeta), both at the protein and the mRNA level, were determined in the left and right ventricle. Left ventricular hypertrophy developed rapidly as early as 1 day after aortic banding followed by further progression at day 7 and day 30. This was paralleled by an increased total PKC enzyme activity in the cytosol fraction and a selectively enhanced protein expression of PKC-delta (day 7, 267+/-18%; day 30, 289+/-12%) and PKC-alpha (day 7, 212+/-20%; day 30, 193+/-14%). The protein amount of PKC- epsilon was not changed in either group. This differential protein expression was associated with a significant increase of the absolute mRNA levels for PKC-delta and PKC-alpha up to 202+/-20% (day 30) and 177+/-17% (day 30), whereas significant alterations in the PKC- epsilon mRNA levels were not detected. A selective upregulation of PKC-alpha and PKC-delta, both on the protein and on the mRNA level, was also noted in the right ventricle during the development of right ventricular hypertrophy, suggesting an adaptive response following elevated left ventricular enddiastolic pressure after long-term aortic banding for 30 days. This study characterizes in the right and left ventricle a differential regulation of the dominant PKC isozymes in pressure-overload cardiac hypertrophy both at the protein and the mRNA level.

Dibutyltin dichloride modifies amylase release from isolated rat pancreatic acini.

Dibutyltin dichloride (DBTC) is widely used as a stabilizer for polyvinylchloride plastics and is of particular toxicologic interest. To examine the effects of DBTC on pancreatic exocrine function in isolated rat pancreatic acini. Isolated rat pancreatic acini were incubated with various secretagogues in the presence or absence of DBTC. We investigated the effects of DBTC on amylase release, receptor binding, and protein kinase C (PKC) enzyme activity. DBTC reduced cholecystokinin octapeptide (CCK-8)-stimulated and carbamylcholine-stimulated amylase release and the binding of [(125)I]CCK-8 to isolated rat pancreatic acini. Conversely, DBTC potentiated secretin-stimulated amylase release, although it slightly inhibited [(125)I]secretin binding to its receptors. In addition, DBTC potentiated amylase release stimulated by vasoactive intestinal peptide, 8-bromoadenosine 3', 5'-monophosphate (8Br-cAMP) or calcium ionophore A23187, whereas it had no influence on amylase release stimulated by 12-O-tetradecanoylphorbol 13-acetate. The protein kinase C (PKC) inhibitor calphostin C abolished the DBTC-induced potentiation of amylase release stimulated by 8Br-cAMP or A23187. Moreover, DBTC caused a significant translocation of PKC enzyme activity from cytosol to membrane fraction. These results indicate that DBTC reduces CCK-8- and carbamylcholine-stimulated amylase release by inhibiting their receptor bindings to pancreatic acini, whereas it potentiates cAMP-mediated amylase release by activating PKC in isolated rat pancreatic acini.

Protein Kinase C Does Not Mediate the Inhibitory Action of Lead on Vitamin D3-Dependent Production of Osteocalcin in Osteoblastic Bone Cells

The level of osteocalcin in serum is lower in lead-intoxicated children than in their normal counterparts. To explain this clinical observation, we investigated the mechanism of action of lead on vitamin D3-dependent osteocalcin production. Lead (5–20 μM) blocked the stimulating effects of vitamin D3 on osteocalcin production in cultured rat osteosarcoma cells (ROS 17/2.8). It is often suggested that activation of protein kinase C (PKC) is a critical mediator of the toxic actions of lead. Treatment of ROS cells with Gö6976, an inhibitor of PKC α and β isozymes, produced similar effects as lead on vitamin D3-dependent osteocalcin production, while activation of PKC by phorbol-12-myristate-13-acetate (TPA) did not reverse or mimic this effect of lead. Thus activation of PKC is not consistent with the actions of lead on vitamin D3-dependent osteocalcin production. Measurement of PKC enzyme activity showed that 10 μM lead treatment does not activate or inhibit the activity of PKC in ROS cells. Western blot analysis indicated that lead treatment does not translocate PKC α, β, or ζ from cytosol to membrane. Therefore, we concluded that PKC does not mediate the cellular toxicity of lead on vitamin D3-dependent osteocalcin production.

Inorganic lead stimulates DNA synthesis in human astrocytoma cells: role of protein kinase Calpha.

As lead has been shown to activate protein kinase C (PKC), and gliomas are reported to be highly dependent on PKC for their proliferation, this study was undertaken to investigate whether lead may act as a mitogen in human astrocytoma cells, and to determine the role of PKC in this effect. Lead acetate (from 100 nM to 100 microM) induced a concentration- and time-dependent increase in DNA synthesis, as measured by incorporation of [methyl-3H]thymidine into cell DNA, without causing any cytotoxicity. Flow cytometric analysis showed that lead was able to stimulate the cell cycle transition from the G0/G1 phase to the S/G2 phase, resulting in increased percentage of cells in the latter phase. Western blot analyses showed that lead induced translocation of PKCalpha, but not of PKCepsilon or PKCzeta, from the cytosolic to the particulate fraction, with a concomitant increase in PKC enzyme activity. Prolonged exposure to lead caused down-regulation of PKCalpha, but not of PKCepsilon. The effect of lead on DNA synthesis was mediated through PKC as evidenced by the finding that two PKC inhibitors, GF 109203X and staurosporine, as well as down-regulation of PKC through prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate, blocked lead-induced DNA synthesis. Further experiments using a pseudosubstrate peptide targeting classical PKCs and selective down-regulation of specific PKC isoforms indicated that the effect of lead on DNA synthesis was mediated by PKCalpha. Altogether, these results suggest that lead stimulates DNA synthesis in human astrocytoma cells by a mechanism that involves activation of PKCalpha.