Protein kinase C isoforms as a target for manic-like behaviors and oxidative stress in a dopaminergic animal model of mania.

Bipolar disorder (BD) is a chronic condition characterized by severe mood swings alternating between episodes of mania and depression. Evidence indicates that protein kinase C (PKC) and oxidative stress are important therapeutic targets for BD. However, what PKC isoforms that are precisely involved in this effect are unknown. Therefore, we evaluated the effects of the intracerebroventricular (ICV) injection of PKC inhibitors (lithium (Li), tamoxifen (TMX), PKCα inhibitor (iPKCα), PKCγ inhibitor (iPKCγ), and PKCε inhibitor (iPKCε)) on the manic-like behaviors and oxidative stress parameters (4-hydroxy-2-nonenal (4-HNE), 8-isoprostane (8-ISO), carbonyl groups, 3-nitrotyrosine (3-NT), glutathione peroxidase (GPx) and glutathione reductase (GR)) in the brains of rats submitted to the model of mania induced by methamphetamine (m-AMPH). Animals received a single ICV infusion of artificial cerebrospinal fluid, Li, TMX, iPKCα, iPKCγ or iPKCε followed by an intraperitoneal injection of saline or m-AMPH before the behavioral analysis (open-field task). Oxidative stress was evaluated in the striatum, frontal cortex, and hippocampus. ICV injection of Li, TMX or iPKCε blocked the m-AMPH-induced increase in the manic-like behaviors - crossings, rearings, visits to the center, sniffing, and grooming. ICV infusion of iPKCα triggered a decrease in these behaviors induced by m-AMPH. Besides, the iPKCε administration significantly prevented the oxidative damage to lipids and proteins, as well as disturbances in the activity of antioxidant enzymes induced by m-AMPH. The findings of the present study suggest that PKCε isoform is strongly implied in the antimanic and antioxidant effects of Li, TMX, and the other PKC inhibitors in the model of mania.

CR4056, a powerful analgesic imidazoline-2 receptor ligand, inhibits the inflammation-induced PKCε phosphorylation and membrane translocation in sensory neurons.

BACKGROUND AND PURPOSE: CR4056 is a first-in-class imidazoline-2 (I2 ) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons. EXPERIMENTAL APPROACH: Effects of CR4056 on bradykinin-induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant-treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals. KEY RESULTS: CR4056 inhibited PKCε translocation with very rapid and long-lasting activity. CR4056 decreased hyperalgesia and phospho-PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved Gi proteins. The inhibition of PKCε translocation by CR4056 was independent of the α2 -adrenoeceptor and, surprisingly, was also independent of idazoxan-sensitive I2 binding sites. The I2 agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I2 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I2 receptor ligand.

Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1ß in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.

miR-205 enhances radiation sensitivity of prostate cancer cells by impairing DNA damage repair through PKCε and ZEB1 inhibition.

BACKGROUND: Radiotherapy is one of the main treatment options for non-metastatic prostate cancer (PCa). Although treatment technical optimization has greatly improved local tumor control, a considerable fraction of patients still experience relapse due to the development of resistance. Radioresistance is a complex and still poorly understood phenomenon involving the deregulation of a variety of signaling pathways as a consequence of several genetic and epigenetic abnormalities. In this context, cumulative evidence supports a functional role of microRNAs in affecting radioresistance, suggesting the modulation of their expression as a novel radiosensitizing approach. Here, we investigated for the first time the ability of miR-205 to enhance the radiation response of PCa models. METHODS: miR-205 reconstitution by a miRNA mimic in PCa cell lines (DU145 and PC-3) was used to elucidate miR-205 biological role. Radiation response in miRNA-reconstituted and control cells was assessed by clonogenic assay, immunofluorescence-based detection of nuclear γ-H2AX foci and comet assay. RNAi was used to silence the miRNA targets PKCε or ZEB1. In addition, target-protection experiments were carried out using a custom oligonucleotide designed to physically disrupt the pairing between the miR-205 and PKCε. For in vivo experiments, xenografts generated in SCID mice by implanting DU145 cells stably expressing miR-205 were exposed to 5-Gy single dose irradiation using an image-guided animal micro-irradiator. RESULTS: miR-205 reconstitution was able to significantly enhance the radiation response of prostate cancer cell lines and xenografts through the impairment of radiation-induced DNA damage repair, as a consequence of PKCε and ZEB1 inhibition. Indeed, phenocopy experiments based on knock-down of either PKCε or ZEB1 reproduced miR-205 radiosensitizing effect, hence confirming a functional role of both targets in the process. At the molecular level, miR-205-induced suppression of PKCε counteracted radioresistance through the impairment of EGFR nuclear translocation and the consequent DNA-PK activation. Consistently, disruption of miR-205-PKCε 3'UTR pairing almost completely abrogated the radiosensitizing effect. CONCLUSIONS: Our results uncovered the molecular and cellular mechanisms underlying the radiosensitizing effect of miR-205. These findings support the clinical interest in developing a novel therapeutic approach based on miR-205 reconstitution to increase PCa response to radiotherapy.

PKCε Increases Extracellular Elastin and Fibulin-5/DANCE in Dermal Fibroblasts.

BACKGROUND/AIMS: In the earlier study, the selective PKCε activator DCP-LA increased elastic fibres in the dermis of HR-1 hairless mice. As a process of elastic fibre formation, tropoelastin, an elastin monomer, is secreted into the extracellular space. Secreted tropoelastin is delivered to the microfibrils by fibulin-5/developmental arteries and neural crest epidermal growth factor-like (DANCE) and undergoes self-association. Then, tropoelastin assembles around the microfibrils, growing into elastin and elastic fibres by lysyl oxidase (LOX)- or LOX-like (LOXL)-mediated cross-linking. The present study was conducted to understand the mechanism underlying DCP-LA-induced increase in elastin/elastic fibre. METHODS: Western blotting, immunocytochemistory, and real-time reverse transcription-polymerase chain reaction (RT-PCR) were carried out in cultured human dermal fibroblasts. PKCε, mammalian target of rapamycin complex (mTOR), and p70 S6 kinase (S6K) were knocked-down by transfecting each siRNA. RESULTS: DCP-LA increased elastin and fibulin-5/DANCE in a treatment time (6-24 h)- and a bell-shaped concentration (1 nM-1 µM)-dependent manner in the culture medium of human dermal fibroblasts. DCP-LA markedly increased elastic fibres in the extracellular space of cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was abolished by a PKC inhibitor or knocking-down PKCε. DCP-LA did not affect expression of mRNAs for tropoelastin and fiblin-5/DANCE in cultured fibroblasts. DCP-LA-induced increase in extracellular elastin and fibulin-5/DANCE was not inhibited by the protein synthesis inhibitor cycloheximide or by knocking-down mTOR and S6K. DCP-LA never increased extracellular elastin in the presence of elastase, that breaks down elastin. An inhibitor of matrix metalloproteinase 9, that degrades multiple extracellular matrix components including elastin, had no effect on the basal levels and the DCP-LA-induced increase levels of extracellular elastin. CONCLUSION: The results of the present study indicate that PKCε, activated by DCP-LA, increases elastin and fibulin-5/DANCE in the extracellular space of cultured fibroblasts by the mechanism independent of transcriptional and translational modulation or inhibition of elastolysis.

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes.

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation.

Angiotensin II-preconditioning is associated with increased PKCε/PKCδ ratio and prosurvival kinases in mitochondria.

Angiotensin II-preconditioning (APC) has been shown to reproduce the cardioprotective effects of ischaemic preconditioning (IPC), however, the molecular mechanisms mediating the effects of APC remain unknown. In this study, Langendorff-perfused rat hearts were subjected to IPC, APC or both (IPC/APC) followed by ischaemia-reperfusion (IR), to determine translocation of PKCε, PKCδ, Akt, Erk1/2, JNK, p38 MAPK and GSK-3ß to mitochondria as an indicator of activation of the protein kinases. In agreement with previous observations, IPC, APC and IPC/APC increased the recovery of left ventricular developed pressure (LVDP), reduced infarct size (IS) and lactate dehydrogenase (LDH) release, compared to controls. These effects were associated with increased mitochondrial PKCε/PKCδ ratio, Akt, Erk1/2, JNK, and inhibition of permeability transition pore (mPTP) opening. Chelerythrine, a pan-PKC inhibitor, abolished the enhancements of PKCε but increased PKCδ expression, and inhibited Akt, Erk1/2, and JNK protein levels. The drug had no effect on the APC- and IPC/APC-induced cardioprotection as previously reported, but enhanced the post-ischaemic LVDP in controls. Losartan, an angiotensin II type 1 receptor (AT1-R) blocker, abolished the APC-stimulated increase of LVDP and reduced PKCε, Akt, Erk1/2, JNK, and p38. Both drugs reduced ischaemic contracture and LDH release, and abolished the inhibition of mPTP by the preconditioning. Chelerythrine also prevented the reduction of IS by APC and IPC/APC. These results suggest that the cardioprotection induced by APC and IPC/APC involves an AT1-R-dependent translocation of PKCε and survival kinases to the mitochondria leading to mPTP inhibition. In chelerythrine-treated hearts, however, alternate mechanisms appear to maintain cardiac function.

Protein kinase C ϵ stabilizes ß-catenin and regulates its subcellular localization in podocytes.

Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of ß-catenin, which promotes its degradation. ß-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with ß-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower ß-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, ß-catenin showed a reversed subcellular localization pattern: Although ß-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates ß-catenin expression and stabilization in a glycogen synthase kinase 3ß-independent manner. Further, ß-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the ß-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in ß-catenin that are required for full ß-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and ß-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of ß-catenin levels and define PKCϵ as an important ß-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes.

RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.

Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11.

PKC-epsilon and TLR4 synergistically regulate resistin-mediated inflammation in human macrophages.

BACKGROUND AND AIMS: Resistin has been associated with atherosclerotic inflammation and cardiovascular complications. We and others have previously shown that PKC-epsilon (PKCε) is involved in resistin-induced smooth muscle cell (VSMC) dysfunction at a high pathological concentration. This study aimed to evaluate the role and potential pathways of resistin at a physiological concentration, in atherosclerosis-related inflammation. METHODS: Plasma from patients with atherosclerosis was analyzed for resistin concentration. Patients were divided into tertiles based on resistin levels and cytokines were compared between tertiles. Macrophages were then treated with resistin in the presence or absence of PKCε inhibitor and/or TLR4 blocking-antibody, and their inflammatory state was evaluated with ELISA, RT-PCR, immunocytochemistry, and Western blot. RESULTS: We observed significant associations between plasma resistin levels and TNF-α, IL-6, IL-12, MIP-1α, MIP-1ß, and CD40L. Our in vitro analyses revealed that resistin activated PKCε via TLR4. This was followed by NF-kB activation and induction of a pro-inflammatory phenotype in macrophages, significantly upregulating CD40, downregulating CD206 and stimulating gene expression and secretion of the inflammatory cytokines, for which we found association in our plasma analysis. Resistin also induced persistent TRAM and CD40L upregulation up to 36 h after resistin treatment. PKCε and TLR4 inhibitors suppressed gene expression to levels similar to control, especially when used in combination. CONCLUSIONS: Resistin, at a physiological concentration, exacerbates the inflammatory response of macrophages. PKCε is a key upstream mediator in resistin-induced inflammation that may interact synergistically with TLR4 to promote NF-kB activation, while TRAM is an important signal. PKCε and TRAM may represent novel molecular targets for resistin-associated chronic atherosclerotic inflammation.

Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen).

Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients.

PKCɛ switches Aurora B specificity to exit the abscission checkpoint.

The 'NoCut', or Aurora B abscission checkpoint can be activated if DNA is retained in the cleavage furrow after completion of anaphase. Checkpoint failure leads to incomplete abscission and a binucleate outcome. These phenotypes are also observed after loss of PKCɛ in transformed cell models. Here we show that PKCɛ directly modulates the Aurora B-dependent abscission checkpoint by phosphorylating Aurora B at S227. This phosphorylation invokes a switch in Aurora B specificity, with increased phosphorylation of a subset of target substrates, including the CPC subunit Borealin. This switch is essential for abscission checkpoint exit. Preventing the phosphorylation of Borealin leads to abscission failure, as does expression of a non-phosphorylatable Aurora B S227A mutant. Further, depletion of the ESCRT-III component and Aurora B substrate CHMP4C enables abscission, bypassing the PKCɛ-Aurora B exit pathway. Thus, we demonstrate that PKCɛ signals through Aurora B to exit the abscission checkpoint and complete cell division.

Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/ßII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and ß protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, ß and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage.

Phosphorylation of the mitochondrial ATP-sensitive potassium channel occurs independently of PKCε in turtle brain.

Neurons from the western painted turtle (Chrysemys picta bellii) are remarkably resilient to anoxia. This is partly due to a reduction in the permeability of excitatory glutamatergic ion channels, initiated by mitochondrial ATP-sensitive K(+) (mK(+)ATP) channel activation. The aim of this study was to determine if: 1) PKCε, a kinase associated with hypoxic stress tolerance, is more highly expressed in turtle brain than the anoxia-intolerant rat brain; 2) PKCε translocates to the mitochondrial membrane during anoxia; 3) PKCε modulates mK(+)ATP channels at the Thr-224 phosphorylation site on the Kir6.2 subunit; and 4) Thr-224 phosphorylation sensitises mK(+)ATP channels to anoxia. Soluble and mitochondrial-rich particulate fractions of turtle and rat cerebral cortex were isolated and PKCε expression was determined by Western blot, which revealed that turtle cortical PKCε expression was half that of the rat. Following the transition to anoxia, no changes in PKCε expression in either the soluble or particulate fraction of the turtle cortex were observed. Furthermore, incubation of tissue with tat-conjugated activator or inhibitor peptides had no effect on the amount of PKCε in either fraction. However, we observed a 2-fold increase in Thr-224 phosphorylation following 1h of anoxia. The increased Thr-224 phosphorylation was blocked by the general kinase inhibitor staurosporine but this did not affect the latency or magnitude of mK(+)ATP channel-mediated mitochondrial depolarization following anoxia, as indicated by rhodamine-123. We conclude that PKCε does not play a role in the onset of mitochondrial depolarization and therefore glutamatergic channel arrest in turtle cerebral cortex.

Selective chemical genetic inhibition of protein kinase C epsilon reduces ethanol consumption in mice.

Reducing expression or inhibiting translocation of protein kinase C epsilon (PKCε) prolongs ethanol intoxication and decreases ethanol consumption in mice. However, we do not know if this phenotype is due to reduced PKCε kinase activity or to impairment of kinase-independent functions. In this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε catalytic activity reduces ethanol consumption. We generated ATP analog-specific PKCε (AS-PKCε) knock-in mice harboring a point mutation in the ATP binding site of PKCε that renders the mutant kinase highly sensitive to inhibition by 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (1-NA-PP1). Systemically administered 1-NA-PP1 readily crossed the blood brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol consumption by AS-PKCε mice but not by wild type mice lacking the AS-PKCε mutation. These results support the development of inhibitors of PKCε catalytic activity as a strategy to reduce ethanol consumption, and they demonstrate that the AS- PKCε mouse is a useful tool to study the role of PKCε in behavior.

Selective Modulation of Protein Kinase C α over Protein Kinase C ε by Curcumin and Its Derivatives in CHO-K1 Cells.

Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKCε, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKCε to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state.

Cyclosporine-A mimicked the ischemic pre- and postconditioning-mediated cardioprotection in hypertensive rats: Role of PKCε.

Our aim was to assess the action of cyclosporine-A (CsA) against reperfusion injury in spontaneously hypertensive rats (SHR) compared to the effects of ischemic pre- (IP) and postconditioning (IPC), examining the role played by PKCε. Isolated hearts were submitted to the following protocols: IC: 45 min global ischemia (GI) and 1h reperfusion (R); IP: a cycle of 5 min GI and 10 min of R prior to 45 min-GI; and IPC: three cycles of 30s-GI/30s-R at the start of R. Other hearts of the IC, IP and IPC groups received CsA (mitochondrial permeability transition pore inhibitor) or chelerythrine (Che, non-selective PKC inhibitor). Infarct size (IS) was assessed. TBARS and reduced glutathione (GSH) content - as parameters of oxidative damage, the expression of P-Akt, P-GSK-3ß, P-PKCε and cytochrome c (Cyc) release - as an index of mitochondrial permeability and the response of isolated mitochondria to Ca(2+) were also measured. IS similarly decreased in preconditioned, postconditioned and CsA treated heart showing the highest values in the combinations IP+CsA and IPC+CsA. TBARS decreased and GSH was partially preserved after all interventions. The content of P-Akt, P-GSK-3ß and P-PKCε increased in cytosol and decreased in mitochondria after IP and IPC. In CsA treated hearts these enzymes increased in both fractions reaching the highest values. Cyc release was attenuated and the response of mitochondria to Ca(2+) was improved by the interventions. The beneficial effects of IP and IPC were annulled when PKC was inhibited with Che. A PKCε/VDAC association was also detected. These data show that, in SHR, the CsA treatment mimicked and reinforced the cardioprotective action afforded by IP and IPC in which PKCε-mediated attenuation of mitochondrial permeability appears as the main mechanism involved.

Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways.

Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.

Serotonin Receptor 2B Mediates Mechanical Hyperalgesia by Regulating Transient Receptor Potential Vanilloid 1.

Serotonin [5-hydroxytryptamine (5-HT)], an inflammatory mediator, contributes to inflammatory pain. The presence of multiple 5-HT subtype receptors on peripheral and central nociceptors complicates the role of 5-HT in pain. Previously, we found that 5-HT2B/2C antagonist could block 5-HT-induced mechanical hyperalgesia. However, the types of neurons or circuits underlying this effect remained unsolved. Here, we demonstrate that the Gq/11-phospholipase Cß-protein kinase Cε (PKCε) pathway mediated by 5-HT2B is involved in 5-HT-induced mechanical hyperalgesia in mice. Administration of a transient receptor potential vanilloid 1 (TRPV1) antagonist inhibited the 5-HT-induced mechanical hyperalgesia. 5-HT injection enhanced 5-HT- and capsaicin-evoked calcium signals specifically in isolectin B4 (IB4)-negative neurons; signals were inhibited by a 5-HT2B/2C antagonist and PKCε blocker. Thus, 5-HT2B mediates 5-HT-induced mechanical hyperalgesia by regulating TRPV1 function.

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

BACKGROUND: Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction. RESULTS: We use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca(2+), stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3',5'-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion. CONCLUSIONS: Together, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.