Calcium-dependent PKC Research Articles

The auxiliary subunit KCNE1 regulates KCNQ1 channel response to sustained calcium-dependent PKC activation.

The slow cardiac delayed rectifier current (IKs) is formed by KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Decrease of IKs currents either due to inherited mutations or pathological remodeling is associated with increased risk for cardiac arrhythmias and sudden death. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in heart disease and diabetes. Recently, we found that sustained stimulation of the calcium-dependent PKCβII isoform leads to decrease in KCNQ1 subunit membrane localization and KCNQ1/KCNE1 channel activity, although the role of KCNE1 in this regulation was not explored. Here, we show that the auxiliary KCNE1 subunit expression is necessary for channel internalization. A mutation in a KCNE1 phosphorylation site (KCNE1(S102A)) abolished channel internalization in both heterologous expression systems and cardiomyocytes. Altogether, our results suggest that KCNE1(S102) phosphorylation by PKCβII leads to KCNQ1/KCNE1 channel internalization in response to sustained PKC stimulus, while leaving KCNQ1 homomeric channels in the membrane. This preferential internalization is expected to have strong impact on cardiac repolarization. Our results suggest that KCNE1(S102) is an important anti-arrhythmic drug target to prevent IKs pathological remodeling leading to cardiac arrhythmias.

Cadmium induces CCL2 production in glioblastoma cells via activation of MAPK, PI3K, and PKC pathways

Cadmium (Cd) is accumulated in human astrocytes and induces the production of interleukin (IL)-6 and IL-8. Astrocytes are one of the major sources of chemokine C–C motif ligand 2 (CCL2; known as monocyte chemoattractant protein-1 [MCP-1]), in the brain. Elevated CCL2 levels are associated with cognitive impairment as well as the migration and invasion of glioblastoma cells. The present study hypothesized that non-toxic concentrations of Cd (as cadmium chloride [CdCl2]) could up-regulate CCL2 production in U-87 MG human glio-blastoma cells. The results showed that after exposure of the U-87 MG cells to CdCl2 at 1 and 10 µM, there was an up-regulation of CCL2 mRNA expression after 3 h of exposure and increased CCL2 secretion after 6 and 24 h. The study also found that inhibition of MAPK pathways, including ERK1/2, p38, and JNK by U0126, SB203580 and SP600125, respectively, reduced Cd-induced CCL2 secretion by the cells. Moreover, when cells were pretreated with Ro 32-0432 (an inhibitor of calcium-dependent PKC) and LY294002 (a PI3K inhibitor), this also resulted in a down-regulation of any Cd-induced CCL2 expression. Taken together, the results of this study allow for the conclusion to be made that CCL2 up-regulation in U-87 MG cells induced by Cd is mediated, in part, by an activation of MAPK, PI3K/Akt, and PKC pathways.

PKC signaling contributes to chromatin decondensation and is required for competence to respond to IL-2 during T cell activation

The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naïve (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.

KCNE1 Subunit Phosphorylation Leads to IKs Internalization in Response to Chronic Calcium-Dependent PKC Activation

Decrease of the cardiac slowly activating delayed rectified K+ current (IKs), formed by KCNQ1 and KCNE1 subunits, is observed in a number of diseases states such as heart failure and diabetes and is associated with increased risk for cardiac arrhythmias and sudden death. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in many pathological conditions. Recently, we found that sustained cPKC stimulation leads to KCNQ1 subunit internalization and decrease of IKs channel activity. Here, we show that KCNE1 is internalized together with KCNQ1 in response to sustained cPKC stimulus using immunostaining and confocal imaging. In addition, we show that KCNE1 is necessary for channel internalization independently of subunit stoichiometry both in heterologous expression systems and cardiomyocytes. Mutation of a PKC phosphorylation site shown to be important to cPKC acute regulation of the channel (KCNE1-S102A) was necessary to channel internalization. Altogether, our results suggest that KCNE1(S102) phosphorylation by cPKC leads to KCNQ1/KCNE1 internalization in response to sustained cPKC stimulus. Our results suggest that KCNE1(S102) is an important target for anti-arrhythmic drugs that would prevent IKs pathological remodeling leading to cardiac arrhythmias.

A calcium-dependent PKC, independent of NF-κB, is required for proper initiation of chromatin decondensation during T-cell activation.

Abstract Following antigen engagement at the T-cell receptor (TCR), there are two known signaling pathways that are involved in the activation of T-cells, initiated by DAG and IP3. These signaling pathways ultimately lead to chromatin decondensation, which is required for subsequent T-cell proliferation mediated by IL-2. It has previously been demonstrated that intracellular calcium is necessary for proper chromatin decondensation but that calcium does not act through transcription factor NFAT. In this study, we demonstrate that PKC, activated through DAG, is also required for proper chromatin decondensation. Furthermore, we show that calcium is required for PKC mediated chromatin decondensation, suggesting that a calcium dependent isoform of PKC is involved in the process. Interestingly, PKC but not IP3 is required for subsequent competence of T-cells to respond to IL-2 signaling. Finally, we show that PKC does not act downstream through NF-κB to decondense chromatin and our data suggest that NF-κB is not required for IL-2 competence. Collectively, these data could be suggestive of multiple mechanisms to control the activation and proliferation of T-cells during an immune response.

Calcium-dependent PKC is required for chromatin decondensation during the activation of peripheral T cells (IRM6P.651)

Abstract Activation of the T cell receptor (TCR) through antigen binding results in chromatin decondensation required for the clonotypic proliferation observed during a proper immune response. Activation of the TCR results in the cleavage of phospholipase biphosphate (PIP2) into diacylglyercol (DAG) and inositol trisphosphate (IP3) by phospholipase C-γ (PLC-γ). Previous studies have shown that both the DAG and IP3 pathways can decondense chromatin, and that intracellular calcium is required for proper TCR-mediated chromatin decondensation. In this study, we used flow cytometry to analyze histone accessibility of chromatin in T cells and found that PKC activation is also required for proper activation-induced chromatin decondensation. Furthermore, we show that calcium is required for proper PKC-mediated chromatin decondensation. Our studies suggest that the PKC isoform(s) required for decondensation may be calcium-dependent. Finally, we show that activation of PKC and not IP3 is required for T cells to gain competence to respond to cytokine stimulation. Taken together, our data suggest that IP3 and PKC signaling work cooperatively to decondense chromatin and regulate clonotypic expansion of T cells during an immune response.

Impaired IKs channel activation by Ca2 +-dependent PKC shows correlation with emotion/arousal-triggered events in LQT1

BackgroundThe most common inherited cardiac arrhythmia, LQT1, is due to IKs potassium channel mutations and is linked to high risk of adrenergic-triggered cardiac events. We recently showed that although exercise-triggered events are very well treated by ß-blockers for these patients, acute arousal-triggered event rate were not significantly reduced after beta-blocker treatment, suggesting that the mechanisms underlying arousal-triggered arrhythmias may be different from those during exercise. IKs is strongly regulated by β-adrenergic receptor (β-AR) signaling, but little is known about the role of α1-AR-mediated regulation. Methods and resultsHere we show, using a combination of cellular electrophysiology and computational modeling, that IKs phosphorylation and α1-AR regulation via activation of calcium-dependent PKC isoforms (cPKC) may be a key mechanism to control channel voltage-dependent activation and consequently action potential duration (APD) in response to adrenergic-stimulus. We show that simulated mutation-specific combined adrenergic effects (β+α) on APD were strongly correlated to acute stress-triggered cardiac event rate for patients while β-AR effects alone were not. ConclusionWe were able to show that calcium-dependent PKC signaling is key to normal QT shortening during acute arousal and when impaired, correlates with increased rate of sudden arousal-triggered cardiac events. Our study suggests that the acute α1-AR-cPKC regulation of IKs is important for QT shortening in “fight-or-flight” response and is linked to decreased risk of sudden emotion/arousal-triggered cardiac events in LQT1 patients.

Protein kinase C delta mediated cytotoxicity of 6-Hydroxydopamine via sustained extracellular signal-regulated kinase 1/2 activation in PC12 cells

Objectives: The incidence of Parkinson's disease (PD) is increasing as the global population ages. 6-hydroxydopamine (6-OHDA) can induce PD-like neuropathology and biochemical changes in both in vitro and in vivo models. Therefore, clarification of the molecular mechanism of 6-OHDA-induced cell death might contribute to the understanding of the pathogenesis of PD.Methods: With this goal in mind, we investigated the role of protein kinase C delta (PKC delta) in 6-OHDA-dependent death using the pheochromocytoma cell line, PC12. Cells were treated with 6-OHDA to induce toxicity with or without pretreatment using rottlerin (a PKC delta inhibitor), bisindolylmaleimide I (a general PKC inhibitor), Gö6976 (a PKC inhibitor selective for calcium-dependent PKC isoforms), or phorbol-12-myristate-13-acetate (PMA, a PKC activator).Results: Phorbol-12-myristate-13-acetate decreased cell survival and increased the rate of apoptosis while rottlerin increased cell survival and decreased the rate of apoptosis. In contrast, neither bisindolylmaleimide I nor Gö6976 affected 6-OHDA-induced cell death. Western analysis demonstrated that phosphorylation of PKC delta on Thr 505 as well as extracellular signal-regulated kinase (ERK) phosphorylation increased after exposure to 6-OHDA. This increase in PKC delta phosphorylation was potentiated by PMA. However, rottlerin attenuated the 6-OHDA-stimulated increase in PKC delta and ERK phosphorylation.Conclusion: These data suggest that PKC delta, rather than classic-type PKC (alpha, beta1, beta2, gamma), participates in 6-OHDA-induced neurotoxicity in PC12 cells, and PKC delta activity is required for subsequent ERK activation during cell death.

Adaptive Regulation Maintains Posttetanic Potentiation at Cerebellar Granule Cell Synapses in the Absence of Calcium-Dependent PKC

Posttetanic potentiation (PTP) is a transient, calcium-dependent increase in the efficacy of synaptic transmission following elevated presynaptic activity. The calcium-dependent protein kinase C (PKC(Ca)) isoforms PKCα and PKCβ mediate PTP at the calyx of Held synapse, with PKCβ contributing significantly more than PKCα. It is not known whether PKC(Ca) isoforms play a conserved role in PTP at other synapses. We examined this question at the parallel fiber → Purkinje cell (PF→PC) synapse, where PKC inhibitors suppress PTP. We found that PTP is preserved when single PKC(Ca) isoforms are knocked out and in PKCα/β double knock-out (dko) mice, even though in the latter all PKC(Ca) isoforms are eliminated from granule cells. However, in contrast to wild-type and single knock-out animals, PTP in PKCα/β dko animals is not suppressed by PKC inhibitors. These results indicate that PKC(Ca) isoforms mediate PTP at the PF→PC synapse in wild-type and single knock-out animals. However, unlike the calyx of Held, at the PF→PC synapse either PKCα or PKCβ alone is sufficient to mediate PTP, and if both isoforms are eliminated a compensatory PKC-independent mechanism preserves the plasticity. These results suggest that a feedback mechanism allows granule cells to maintain the normal properties of short-term synaptic plasticity even when the mechanism that mediates PTP in wild-type mice is eliminated.

The coronary circulation in exercise training.

Exercise training (EX) induces increases in coronary transport capacity through adaptations in the coronary microcirculation including increased arteriolar diameters and/or densities and changes in the vasomotor reactivity of coronary resistance arteries. In large animals, EX increases capillary exchange capacity through angiogenesis of new capillaries at a rate matched to EX-induced cardiac hypertrophy so that capillary density remains normal. However, after EX coronary capillary exchange area is greater (i.e., capillary permeability surface area product is greater) at any given blood flow because of altered coronary vascular resistance and matching of exchange surface area and blood flow distribution. The improved coronary capillary blood flow distribution appears to be the result of structural changes in the coronary tree and alterations in vasoreactivity of coronary resistance arteries. EX also alters vasomotor reactivity of conduit coronary arteries in that after EX, α-adrenergic receptor responsiveness is blunted. Of interest, α- and β-adrenergic tone appears to be maintained in the coronary microcirculation in the presence of lower circulating catecholamine levels because of increased receptor responsiveness to adrenergic stimulation. EX also alters other vasomotor control processes of coronary resistance vessels. For example, coronary arterioles exhibit increased myogenic tone after EX, likely because of a calcium-dependent PKC signaling-mediated alteration in voltage-gated calcium channel activity in response to stretch. Conversely, EX augments endothelium-dependent vasodilation throughout the coronary arteriolar network and in the conduit arteries in coronary artery disease (CAD). The enhanced endothelium-dependent dilation appears to result from increased nitric oxide bioavailability because of changes in nitric oxide synthase expression/activity and decreased oxidant stress. EX also decreases extravascular compressive forces in the myocardium at rest and at comparable levels of exercise, mainly because of decreases in heart rate and duration of systole. EX does not stimulate growth of coronary collateral vessels in the normal heart. However, if exercise produces ischemia, which would be absent or minimal under resting conditions, there is evidence that collateral growth can be enhanced. While there is evidence that EX can decrease the progression of atherosclerotic lesions or even induce the regression of atherosclerotic lesions in humans, the evidence of this is not strong due to the fact that most prospective trials conducted to date have included other lifestyle changes and treatment strategies by necessity. The literature from large animal models of CAD also presents a cloudy picture concerning whether EX can induce the regression of or slow the progression of atherosclerotic lesions. Thus, while evidence from research using humans with CAD and animal models of CAD indicates that EX increases endothelium-dependent dilation throughout the coronary vascular tree, evidence that EX reverses or slows the progression of lesion development in CAD is not conclusive at this time. This suggests that the beneficial effects of EX in CAD may not be the result of direct effects on the coronary artery wall. If this suggestion is true, it is important to determine the mechanisms involved in these beneficial effects.

IL-33 augments substance P–induced VEGF secretion from human mast cells and is increased in psoriatic skin

The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1-10 microM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5-100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.

A novel role for the TRPV1 channel in UV‐induced matrix metalloproteinase (MMP)‐1 expression in HaCaT cells

Transient receptor potential vanilloid type 1 (TRPV1) is a molecular sensor for detecting adverse stimuli, such as capsaicin, heat, and acid. TRPV1 has been localized in keratinocytes and is suggested to be a mediator of heat-induced matrix metalloproteinase-1 (MMP-1). With regard to the multimodal activation of TRPV1, we hypothesize that TRPV1 might also mediate UV-induced MMP-1 in keratinocytes. In HaCaT, a human keratinocyte cell line, we initially confirmed capsaicin-induced membrane current and Ca(2+) influx. UV irradiation induced slow and persistent calcium influx and increased membrane current, which was inhibited by TRPV1 inhibitors (capsazepine and ruthenium red). The UV-induced MMP-1 expression in HaCaT was also decreased by TRPV1 inhibitors and was facilitated by capsaicin. Knock-down of TRPV1 using siRNA transfection also decreased MMP-1 expression, as well as UV-induced Ca(2+) influx in HaCaT. UV failed to induce MMP-1 expression in HaCaT cells cultured in Ca(2+)-free media. Both the UV-induced increase in [Ca(2+)](i) and MMP-1 were suppressed by Gö6976 (a calcium-dependent PKC inhibitor), but not by rottlerin (a calcium-independent PKC inhibitor). In addition to a plausible role of TRPV1 in UV-induced MMP-1 expression, we showed that UV increased TRPV1 expression in both HaCaT cells and human skin in vivo. From these results, we suggest that UV-induced MMP-1 expression might be mediated in part by PKC-dependent activation of TRPV1 and subsequent Ca(2+)-influx in human keratinocytes. J. Cell. Physiol. 219: 766-775, 2009. (c) 2009 Wiley-Liss, Inc.

Both Membrane Depolarization And IL-6 Induce Calcium-Dependent Hsp70 Expression In Skeletal Muscle Cells

Adaptive response of skeletal muscle to challenges imposed by contractile activity is associated to changes in specific genes expression. IL-6 and Hsp70 are proteins involved in the maintenance of skeletal muscle homeostasis during stress episodes and are markedly expressed in skeletal muscle after physiological contraction. Muscle-derived IL-6 has systemic and local effects acting in a hormone-like fashion, nevertheless the molecular bases of its functional role on skeletal muscle is poorly understood. We have demonstrated that depolarization evoked IP3 mediated slow calcium transients, associated to cell nuclei are involved in the up-regulation of IL-6 transcriptional activity in skeletal muscle cells.The aim of this work was to investigate calcium involvement in Hsp70 expression in both depolarized and IL-6 treated skeletal muscle cells.We observed that electrical stimulation of myotubes increases Hsp70 mRNA level and protein expression. Depolarization performed in the presence of the intracellular calcium chelator BAPTA-AM resulted in a complete inhibition of Hsp70 induced expression. Inhibitors of IP3-dependent calcium signals like 2-aminoethoxydiphenyl borate (2-APB) and LY294002, decreased Hsp70 mRNA induction and the protein expression in depolarized cells. In addition we determined that inhibitors of calcium dependent PKC abolished Hsp70 mRNA induction.We established that IL-6 treatment of myotubes induced changes in intracellular calcium and promoted the increase of Hsp70 mRNA levels. Observed calcium transients could be associated to early events of IL-6-mediated Hsp70 expression.Our results provide evidence for the involvement of slow calcium transients and PKC in the activation of Hsp70 expression in skeletal muscle cells and suggest that intracellular calcium signals also participate in IL-6 induced Hsp70 expression.FONDAP 15010006

Subversion of CtBP1-controlled macropinocytosis by human adenovirus serotype 3

Endocytosis supports cell communication, growth, and pathogen infection. The species B human adenovirus serotype 3 (Ad3) is associated with epidemic conjunctivitis, and fatal respiratory and systemic disease. Here we show that Ad3 uses dynamin-independent endocytosis for rapid infectious entry into epithelial and haematopoietic cells. Unlike Ad5, which uses dynamin-dependent endocytosis, Ad3 endocytosis spatially and temporally coincided with enhanced fluid-phase uptake. It was sensitive to macropinocytosis inhibitors targeting F-actin, protein kinase C, the sodium-proton exchanger, and Rac1 but not Cdc42. Infectious Ad3 macropinocytosis required viral activation of p21-activated kinase 1 (PAK1) and the C-terminal binding protein 1 of E1A (CtBP1), recruited to macropinosomes. These macropinosomes also contained the Ad3 receptors CD46 and alpha v integrins. CtBP1 is a phosphorylation target of PAK1, and is bifunctionally involved in membrane traffic and transcriptional repression of cell cycle, cancer, and innate immunity pathways. Phosphorylation-defective S147A-CtBP1 blocked Ad3 but not Ad5 infection, providing a direct link between PAK1 and CtBP1. The data show that viruses induce macropinocytosis for infectious entry, a pathway used in antigen presentation and cell migration.

168 MOLECULAR INTERACTION BETWEEN BCL-XL AND BNIP3 DETERMINES THE CELL FATE OF HYPERTROPHIC CHONDROCYTES

demonstrated that siRNA for Ank reduced 4-fold more than siRNA for PC-1 the inducing effect of TGF-β1 on ePPi levels. We showed that neither SB203580 (a p38 MAPK inhibitor), nor RcAMP (a PKA inhibitor) were able to modulate Ank induction, but that PD98059 (a MEK 1 inhibitor) was strongly active, ensuring a 50% inhibition of TGF-β-1-induced Ank expression. On the other hand, the effect of PKC inhibitors depended on their selectivity: PKCδ inhibitor (rottlerin) was weakly effective compared to Ca2+-dependent PKC inhibitor (Go6976), that diminished Ank expression by 60%, suggesting the involvement of calcium-dependent PKC in the regulation of Ank gene by TGF-β1. Finally, we demonstrated with plasmids transfection the involvement of Ras, Raf-1 and confirmed the contribution of ERK in Ank mRNA regulation. However, overexpression of Smad 7 did not affect Ank expression, showing that Smad-dependent signaling events downstream to TGF-β1 were not implicated. Conclusions: These results indicate that TGF-β1 mediates an increase in ePPi concentration, mainly by induction of Ank expression, which depends on activation of Ras, Raf-1, ERK and Ca2+-dependent PKC pathways in chondrocyte. Our results underline the interest to modulate Ras/Raf-1/ERK or PKC signaling pathways in chondrocytes, as it could open insights for the treatment of sporadic chondrocalcinosis.

Spike protein of SARS‐CoV stimulates cyclooxygenase‐2 expression via both calcium‐dependent and calcium‐independent protein kinase C pathways

We have previously shown that the nucleocapsid protein of SARS-associated coronavirus (SARS-CoV) activated cyclooxygenase-2 (COX-2) expression. In this study, we identified another viral protein, the spike of SARS-CoV, which played an important role in virus-stimulated COX-2 expression after screening all genes from the SARS-CoV genome. We found that an upstream calcium-dependent PKC isozyme PKC alpha that modulates the downstream ERK/NF-kappaB pathway through an influx of extracellular Ca2+ is induced by the spike protein of SARS-CoV. The ERK/NF-kappaB was identified to be involved in the activation of COX-2 promoter and production of COX-2 protein in HEK293T cells. We also demonstrated that another unusual pathway, the calcium-independent PI3K/PKC epsilon/JNK/CREB pathway, functioned in cooperation with the calcium-dependent pathway to induce COX-2 expression upon stimulation by spike protein. This pathway can be blocked by PKC epsilon-specific, small interfering RNA, PI3K/JNK kinase-specific inhibitors as well as dominant negative JNK. PKC epsilon-specific siRNA also attenuated the phosphorylation of JNK. Our results provide evidence that helps us understand the function of SRAS-CoV spike protein in SARS pathogenesis.

The identification of a functional interaction between PKC and topoisomerase II

Topoisomerase II plays an essential role in the segregation of chromosomes during cell division. It is also a major component of the nuclear matrix. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between the catalytic domain of the yeast protein kinase 1 enzyme (Pkc1) and the N-terminal domain of the S. cerevisiae topoisomerase II. The S. cerevisiae Pkc1 is the homologue of the mammalian calcium dependent PKC.

A Commercial Mixture of the Brominated Flame Retardant Pentabrominated Diphenyl Ether (DE-71) Induces Respiratory Burst in Human Neutrophil Granulocytes In Vitro

Polybrominated diphenyl ethers (PBDEs) are widely used brominated flame retardants (BFRs), which have become ubiquitous in the environment. This study investigates the effects of the pentabrominated diphenyl ether mixture, DE-71, on human neutrophil granulocytes in vitro. DE-71 enhanced production of reactive oxygen species (ROS) in a concentration-dependent manner measured as lucigenin-amplified chemiluminescence. Octabrominated diphenyl ether (OBDE), decabrominated diphenyl ether (DBDE), and the non-brominated diphenyl ether did not induce ROS formation at the concentrations tested. DPI (4 microM), an inhibitor of the NADPH oxidase completely inhibited DE-71 induced ROS formation, highlighting a role for NADPH oxidase activation. The protein kinase C inhibitor BIM (0.25 microM) and the selective chelator of intracellular calcium, BAPTA-AM (5 microM), also inhibited NADPH oxidase activation, indicating a calcium-dependent activation of PKC. ROS formation was also inhibited by the tyrosine kinase inhibitor tyrphostin (1 microM), the phospholipase C inhibitor ET-18-OCH3 (5 microM), and the phosphatidylinositol-3 kinase inhibitor LY294002 (25 microM). Alterations in intracellular calcium were measured using fura-2/AM, and a significant increase was measured after exposure to DE-71 both with and without extracellular calcium. The tetra brominated compound BDE-47 also enhanced ROS formation in a concentration dependent manner. The combination of DE-71 with the bacteria-derived N-formyl peptide fMLP and PCB153 induced an additive effect in the lucigenin assay. We suggest that tyrosine kinase mediated activation of PI3K could result in enhanced activation of calcium-dependent PKC by enhanced PLC activity, followed by intracellular calcium release leading to ROS formation in neutrophil granulocytes.

TGF‐β induced hyaluronan synthesis in orbital fibroblasts involves protein kinase C βII activation in vitro

Graves' ophthalmopathy is accompanied by hyaluronan (HA) accumulation in the orbital space and infiltration of immunocompetent cells and cytokines, including IFN-gamma, IL-1beta, and TGF-beta. We examined the signal transduction pathways by which TGF-beta induces HA synthesis in normal orbital fibroblasts, orbital fibroblasts from patients with Graves' ophthalmopathy, and abdominal fibroblasts. Calphostin C inhibited the stimulation of HA synthesis by TGF-beta. Phorbol 12-myristate 13-acetate (PMA) activation of PKC stimulated HA production. The effects of TGF-beta and PMA were not synergistic. Stimulation by TGF-beta and PMA were dependent on protein synthesis and their effects were inhibited by cycloheximide. Since TGF-beta-induced HA synthesis was inhibited by BAPTA or by PKC inhibitors, a calcium-dependent PKC was most likely involved. The PKA inhibitor H-89 enhanced TGF-beta- and PMA-induced HA synthesis, thus showing that communication between the PKA and PKC pathways was evident. TGF-beta stimulated the translocation of PKCbetaII to the cell membrane. PKCbetaII, a key enzyme in the regulation of HA synthesis by TGF-beta, might be an appropriate target for therapeutic compounds to be used to treat Graves' ophthalmopathy accompanied by inflammation.

Akt Activation in Platelets Depends on Gi Signaling Pathways

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonist-induced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr308 and Ser473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental Gi or Gz signaling. Unlike wild-type mouse platelets, platelets from Galphaq-deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y1 receptor. However, ADP caused Akt phosphorylation in Galphaq- and P2Y1-deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that Gi, but not Gq or G12/13, signaling pathways are required for activation of Akt in platelets, and Gi signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets.