Negative feedback regulation of Gq signaling by protein kinase C is disrupted by diacylglycerol kinase ζ in COS-7 cells

Cellular response to G(q)-linked agonists is shaped by regulatory inputs which determine signal strength and duration. Stimulation of phospholipase C-β (PLC-β) lipase activity results in an increase in the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC) activity. PKC has been implicated in the feedback regulation of G(q) signaling through actions on PLC-β and phospholipase D (PLD) lipase activity. As PKC activity is modulated by multiple layers of regulation, the physiological impact of PKC on G(q) signaling is unclear. PKC signaling can be terminated by diacylglycerol kinases (DGKs) which are regulated in a cell-specific manner. The present studies investigated the contribution of the ubiquitously expressed DGKζ isoform in the regulation of PKC signaling and G(q) response in transfected COS-7 cells. Genetic depletion of DGKζ protein with antisense oligonucleotides dramatically reduced DAG metabolism. The sustained increase in PKC signaling was associated with a pronounced inhibition of carbachol-stimulated lipase activity in cells co-transfected with m1 muscarinic receptor, Gα(q) and either with or without PLC-β(1). The data also reveal that sustained activation of PKC alone does not increase cellular PLD1 activity. Therefore, G(12)-activated RhoA is physiologically important for adequate stimulation of PLD1 activity. These data show that the impact of PKC on G(q) signal transduction is determined by the background of cell-specific processes.     

Involvement of protein kinase Cδ in induction of apoptosis by cationic liposomes in macrophage-like RAW264.7 cells

We have recently demonstrated that reactive oxygen species (ROS) play an important role in RAW264.7 cell apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes). In this study, we investigated whether protein kinase Cδ PKCδ) is involved in apoptosis induced by cationic liposomes. Tyrosine phosphorylation, nuclear localization, and cleavage of PKCδ were observed following the treatment of cells with SA-liposomes, suggesting that SA-liposomes activate PKCδ. Rottlerin, a specific inhibitor of PKCδ, inhibited ROS generation and also suppressed apoptosis. Cell surface proteoglycans may contribute to PKCδ activation by SA-liposomes. These findings suggest that PKCδ is strongly associated with apoptosis induced by SA-liposomes.     

Overexpression of protein kinase C ? improves retention and survival of transplanted mesenchymal stem cells in rat acute myocardial infarction

We assessed the effects of protein kinase C ɛ (PKCɛ) for improving stem cell therapy for acute myocardial infarction (AMI). Primary mesenchymal stem cells (MSCs) were harvested from rat bone marrow. PKCɛ-overexpressed MSCs and control MSCs were transplanted into infarct border zones in a rat AMI model. MSCs and PKCɛ distribution and expression of principal proteins involved in PKCɛ signaling through the stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor type 4 (CXCR4) axis and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway were analyzed by immunofluorescence and western blot 1 day after transplantation. Echocardiographic measurements and histologic studies were performed at 4 weeks after transplantation, and MSC survival, expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGFβ), cardiac troponin I (cTnI), von Willebrand factor (vWF), smooth muscle actin (SMA) and factor VIII and apoptosis in infarct border zones were assessed. Rat heart muscles retained more MSCs and SDF-1, CXCR4, PI3K and phosphorylated AKT increased with PKCɛ overexpression 1 day after transplantation. MSC survival and VEGF, bFGF, TGFβ, cTnI, vWF, SMA and factor VIII expression increased in animals with PKCɛ-overexpressed MSCs at 4 weeks after transplantation and cardiac dysfunction and remodeling improved. Infarct size and apoptosis decreased as well. Inhibitory actions of CXCR4 or PI3K partly attenuated the effects of PKCɛ. Activation of PKCɛ may improve retention, survival and differentiation of transplanted MSCs in myocardia. Augmentation of PKCɛ expression may enhance the therapeutic effects of stem cell therapy for AMI.Irreversible and widespread loss of myocardial cells and subsequent ventricular remodeling induced by acute myocardial infarction (AMI) is the main cause of chronic heart failure¹ and globally >17 million people died of ischemic heart diseases in 2008.² Stem cell-based regenerative therapy for AMI is encouraging with respect to preclinical^{3, 4} and clinical data,^{5, 6, 7, 8} and this may soon be a therapeutic modality for injury resulting from coronary artery disease. Two problems – poor homing of transplanted cells to injury sites, and poor cell survival – require resolution before transplantation therapy can be broadly effective. The stromal cell-derived factor 1 (SDF-1)/CXC chemokine receptor type 4 (CXCR4) axis has an important role during migration, proliferation and survival of stem cells, but using this knowledge to improve homing and survival of therapeutic stem cells has not been successful.Previous studies^{9, 10, 11} suggest that protein kinase C ɛ (PKCɛ) is essential for signal transduction for ischemic cardioprotection, but whether it has an effect on stem cell retention and survival and what mechanism underlies this effect is uncertain. We know that SDF-1 increased significantly in mesenchymal stem cells (MSCs) after treatment with PKC activator and decreased after treatment with a PKCɛ inhibitor in preliminary experiments, and our latest work indicates that activating PKCɛ improves migration and paracrine function of MSCs in vitro.¹² Thus, we suggest that PKCɛ overexpression in transplanted bone marrow MSCs (BMMSCs) would improve retention and survival of MSC''s and improve cardiac function and remodeling in a rat AMI model.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

The gating of nucleotide-sensitive K+ channels in insulin-secreting cells can be modulated by changes in the ratio ATP4−/ADP3− and by nonhydrolyzable derivatives of both ATP and ADP

Summary The³¹P-NMR technique has been used to assess the intracellular ratios and concentrations of mobile ATP and ADP and the intracellular pH in an insulin-secreting cell line, RINm5F. The single-channel current-recording technique has been used to investigate the effects of changes in the concentrations of ATP and ADP on the gating of nucleotide-dependent K⁺ channels. Adding ATP to the membrane inside closes these channels. However, in the continued presence of ATP adding ADP invariably leads to the reactivation of ATP-inhibited K⁺ channels, even at ATP^4–/ADP^3– concentration ratios greater than 71. Interactions between ATP^4– and ADP^3– seem competitive. An increase in the concentration ratio ATP^4–/ADP^3– consistently evoked a decrease in the open-state probability of K⁺ channels; conversely a decrease in ATP^4–/ADP^3– increased the frequency of K⁺ channel opening events. Channel gating was also influenced by changes in the absolute concentrations of ATP^4– and ADP^3–, at constant free concentration ratios. ADP-evoked stimulation of ATP-inhibited channels did not result from phosphorylation of the channel, as ADP--S, a nonhydrolyzable analog of ADP, not only stimulated but enhanced ADP-induced activation of K⁺ channels, in the presence of ATP. Similarly, ADP was able to activate K⁺ channels in the presence of two nonhydrolyzable derivatives of ATP, AMP-PNP and methylene ATP.     

The localization of the β-subtype of protein kinase C (PKC-β) in rat sympathetic neurons

Summary The localization of PKC- was studied in rat sympathetic neurons using a polyclonal antibody specific for the ₁- and ₂-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC--LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC--LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC--immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC--LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC- in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC- in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.     

Isomers of conjugated linoleic acid induce insulin resistance through a mechanism involving activation of protein kinase Cε in liver cells

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser⁴⁷³ and Thr³⁰⁸, the insulin receptor at Tyr¹¹⁵⁸, IRS-1 at Tyr⁶³², and GSK-3 at Ser^9/21. Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser⁶¹², a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation.     

Colocalization of protein kinase C ⊺-subtype and calcitonin gene-related peptide in rat spinal cord

Summary Colocalization of calcitonin gene-related peptide (CGRP) and protein kinase C -subtype (PKC-) like immunoreactivities (LI) and the subcellular localization of CGRP-LI were studied in the ventral horn of rat spinal cord. Ultrastructurally CGRP-LI was localized on the membranes of the Golgi-complexes, in multivesicular bodies and in vesicles adjacent to the Golgi-complex in motoneuron perikarya. The colocalization of PKC- and CGRP-LI was detected in most of the ventral horn motoneurons. However, few motoneurons were only PKC--immunoreactive. These results suggest that PKC- may be involved in the regulation of CGRP release from motoric axon terminals.     

Role of protein kinase C δ in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells

During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.     

Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cδ, and p38 mitogen-activated protein kinase (MAPK)

Mer tyrosine kinase (MerTK) is an integral membrane protein that is preferentially expressed by phagocytic cells, where it promotes efferocytosis and inhibits inflammatory signaling. Proteolytic cleavage of MerTK at an unidentified site leads to shedding of its soluble ectodomain (soluble MER; sMER), which can inhibit thrombosis in mice and efferocytosis in vitro. Herein, we show that MerTK is cleaved at proline 485 in murine macrophages. Site-directed deletion of 6 amino acids spanning proline 485 rendered MerTK resistant to proteolysis and suppression of efferocytosis by cleavage-inducing stimuli. LPS is a known inducer of MerTK cleavage, and the intracellular signaling pathways required for this action are unknown. LPS/TLR4-mediated generation of sMER required disintegrin and metalloproteinase ADAM17 and was independent of Myd88, instead requiring TRIF adaptor signaling. LPS-induced cleavage was suppressed by deficiency of NADPH oxidase 2 (Nox2) and PKCδ. The addition of the antioxidant N-acetyl cysteine inhibited PKCδ, and silencing of PKCδ inhibited MAPK p38, which was also required. In a mouse model of endotoxemia, we discovered that LPS induced plasma sMER, and this was suppressed by Adam17 deficiency. Thus, a TRIF-mediated pattern recognition receptor signaling cascade requires NADPH oxidase to activate PKCδ and then p38, culminating in ADAM17-mediated proteolysis of MerTK. These findings link innate pattern recognition receptor signaling to proteolytic inactivation of MerTK and generation of sMER and uncover targets to test how MerTK cleavage affects efferocytosis efficiency and inflammation resolution in vivo.     

Tamoxifen depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase Cα in rat cerebral cortex nerve terminals

This study was aimed at examining the effect of tamoxifen, a selective estrogen receptor modulator, on the release of endogenous glutamate in rat cerebral cortex nerve terminals (synaptosomes) and exploring the possible mechanism. Tamoxifen inhibited the release of glutamate that was evoked by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent and insensitive to the estrogen receptor antagonist. The effect of tamoxifen on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate did not have any effect on the action of tamoxifen. Tamoxifen did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in cytosolic [Ca(2+)]. Furthermore, the inhibitory effect of tamoxifen on the evoked glutamate release was abolished by the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na(+)/Ca(2+) exchanger blocker CGP37157. In addition, the protein kinase C (PKC) inhibitors GF109203X or Ro318220 prevented tamoxifen from inhibiting glutamate release. Western blotting showed that tamoxifen significantly decreased the 4-AP-induced phosphorylation of PKC and PKCα. Together, these results suggest that tamoxifen inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca(2+) entry and PKC activity.     

cAMP increases the sensitivity of exocytosis to Ca²+ primarily through protein kinase A in mouse pancreatic beta cells

Cyclic AMP regulates the late step of Ca2+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca2+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca2+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C(m) change had typically two phases. We observed that the Ca2+-dependency of the rate of the first C(m) change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9±0.2 μM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca2+-dependency of the exocytotic burst to significantly lower [Ca2+](i). To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca2+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 μM 6-Phe-cAMP or inhibition with 500 μM Rp-cAMPs in beta cells significantly shifted the EC(50) in the opposite directions. Specific activation of Epac2 did not change Ca2+ sensitivity. Our findings suggest that cAMP modulates Ca2+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca2+](i); Epac2 may contribute to enhance the rates of secretory vesicle fusion.     

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cɛ for their functional regulation in rat brain

Background

We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359–367].

Methods

By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain.

Results

We found that (i) syndapin 1 and novel protein kinase C? (nPKC?) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKC? as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKC? in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3β; (v) syndapin 1 highly stimulated the GSK-3β-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain.

General significance

These results provided here suggest that (i) TP-associated nPKC? suppresses the GSK-3β-mediated phosphorylation of TP through the phosphorylation of GSK-3β by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3β-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.     

Acute regulation of 5′-AMP-activated protein kinase by long-chain fatty acid,glucose and insulin in rat primary adipocytes

Palmitate increased AMPK (5′-AMP-activated protein kinase) activity, glucose utilization and 2-DOG (2-deoxyglucose) transport in rat adipocytes. All three effects were blocked by the AMPK inhibitor Compound C, leading to the conclusion that in response to an increase in long-chain NEFA (non-esterified fatty acid) concentration AMPK mediated an enhancement of adipocyte glucose transport, thereby providing increased glycerol 3-phosphate for FA (fatty acid) esterification to TAG (triacylglycerol). Activation of AMPK in response to palmitate was not due to an increase in the adipocyte AMP:ATP ratio. Glucose decreased AMPK activity and effects of palmitate and glucose on AMPK activity were antagonistic. While insulin had no effect on basal AMPK activity insulin did decrease AMPK activity in the presence of palmitate and also decreased the percentage effectiveness of palmitate to increase the transport of 2-DOG. It is suggested that activation of adipocyte AMPK by NEFA, as well as decreasing the activity of hormone-sensitive lipase, could modulate adipose tissue dynamics by increasing FA esterification and, under certain circumstances, FA synthesis.     

Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin‐releasing hormone (GnRH): Involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin‐releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH‐R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real‐time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LβT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)‐C pathway, exposure of the cells to phorbol 12,13‐dibutyrate rapidly elevates both mPer1 and LHβ subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHβ response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen‐activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH‐induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.