Inhibition Of Protein Kinase C Activity Research Articles

In vivo study of a novel protein kinase C that mediates immunocompetence and catecholamine biosynthesis in hemocytes of Litopenaeus vannamei by using its potential competitive inhibitor, bisindolylmaleimide I

This study applied bisindolylmaleimide I (BSM), a pharmacological competitive inhibitor of protein kinase C (PKC) enzymatic activity, at 1.25 pmol shrimp−1 for 60 min to investigate the potential involvement of PKC in signal transduction pathways in the hemocytes of Litopenaeus vannamei. A novel PKC in L. vannamei (LvnPKC) was identified and characterized and was determined to be involved in mediating the neuroendocrine-immune regulatory network. The hemocytes of L. vannamei that receive BSM exhibit significantly decreased PKC activity and LvnPKC gene and protein expression levels. Furthermore, the total hemocyte count, hyaline cells, and semigranular cells increased significantly along with significant decreases in granular cells, and meanwhile, the significantly increased phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and neutrophil extracellular trap were observed; however, phagocytic activity decreased significantly. In a molecular model, the gene expressions of lipopolysaccharide- and β-1,3-glucan-binding protein, peroxinectin, cytosolic manganese SOD, mitochondrial manganese SOD, and copper/zinc SOD in the hemocytes of L. vannamei that had received BSM decreased significantly, but prophenoloxidase I increased significantly. In catecholamine biosynthesis, tyrosine, dopamine, and norepinephrine decreased significantly in the hemocytes of L. vannamei that had received BSM, and l-dihydroxyphenylalanine increased. Moreover, tyrosine hydroxylase (TH) activity increased significantly, whereas TH and dihydroxyphenylalanine decarboxylase gene expression decreased significantly. These findings suggest that BSM inhibits PKC activity in hemocytes in which LvnPKC gene and protein expression are also inhibited. Additionally, the hemocytes’ immunocompetence, including their prophenoloxidase and antioxidant systems, phagocytic activity, and catecholamine biosynthesis, was disrupted, confirming the roles of LvnPKC in mediating the neuroendocrine-immune regulatory network in hemocytes.

Trinuclear Calcium Site in the C2 Domain of PKCα/γ Is Prone to Lithium Attack.

Lithium (Li+) is the first-line therapy for bipolar disorder and a candidate drug for various diseases such as amyotrophic lateral sclerosis, multiple sclerosis, and stroke. Despite being the captivating subject of many studies, the mechanism of lithium’s therapeutic action remains unclear. To date, it has been shown that Li+ competes with Mg2+ and Na+ to normalize the activity of inositol and neurotransmitter-related signaling proteins, respectively. Furthermore, Li+ may co-bind with Mg2+-loaded adenosine or guanosine triphosphate to alter the complex’s susceptibility to hydrolysis and mediate cellular signaling. Bipolar disorder patients exhibit abnormally high cytosolic Ca2+ levels and protein kinase C (PKC) hyperactivity that can be downregulated by long-term Li+ treatment. However, the possibility that monovalent Li+ could displace the bulkier divalent Ca2+ and inhibit PKC activity has not been considered. Here, using density functional theory calculations combined with continuum dielectric methods, we show that Li+ may displace the native dication from the positively charged trinuclear site in the C2 domain of cytosolic PKCα/γ. This would affect the membrane-docking ability of cytosolic PKCα/γ and reduce the abnormally high membrane-associated active PKCα/γ levels, thus downregulating the PKC hyperactivity found in bipolar patients.

Anti-Malignant Ascites Effect of Total Diterpenoids from Euphorbiae Ebracteolatae Radix Is Attributable to Alterations of Aquaporins via Inhibiting PKC Activity in the Kidney.

This study evaluated the anti-ascites effect of total diterpenoids extracted from Euphorbiae ebracteolatae Radix (TDEE) on malignant ascitic mice and elucidated its underlying mechanism. TDEE was extracted by dichloromethane and subjected to column chromatography. The purity of six diterpenoids isolated from TDEE was determined to be 77.18% by HPLC. TDEE (3 and 0.6 g raw herbs/kg, p.o.) reduced ascites and increased urine output. Meanwhile, analysis of tumor cell viability, cycle and apoptosis indicated that TDEE had no antitumor activity. In addition, the expression levels of aquaporins (AQPs) and the membrane translocation levels of protein kinase C (PKC) α and PKCβ in kidney and cells were measured. TDEE reduced the levels of AQP1–4, and inhibited PKCβ expression in membrane fraction. Four main diterpenoids, except compound 2, reduced AQP1 level in human kidney-2 cells. Compounds 4 and 5 inhibited AQP2–4 expression in murine inner medullary collecting duct cells. The diterpenoid-induced inhibition of AQP1–4 expression was blocked by phorbol-12-myristate-13-acetate (PMA; agonist of PKC). The diterpenoids from TDEE are the main anti-ascites components. The anti-ascites effect of diterpenoids may be associated with alterations in AQPs in the kidneys to promote diuresis. The inhibition of AQP1–4 expression by TDEE is related to the inhibition of PKCβ activation.

Cudrania tricuspidata Root Extract Prevents Methylglyoxal-Induced Inflammation and Oxidative Stress via Regulation of the PKC-NOX4 Pathway in Human Kidney Cells.

Diabetic nephropathy is a microvascular complication induced by diabetes, and methylglyoxal (MGO) is a reactive carbonyl species causing oxidative stress that contributes to the induction of inflammatory response in kidney cells. Cudrania tricuspidata (CT), cultivated in Northeast Asia, has been used as traditional medicine for treating various diseases, including neuritis, liver damage, and cancer. In this study, we determined whether a CT root extract (CTRE) can prevent MGO-induced reactive oxygen species (ROS) production and inflammation and assessed underlying mechanisms using a kidney epithelial cell line, HK-2. We observed that CTRE inhibited MGO-induced ROS production. Additionally, CTRE ameliorated the activation of MGO-induced inflammatory signaling pathways such as p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-JUN N-terminal kinase (JNK). Consistent with these results, expressions of p-nuclear factor-kappa B (NFκB) and inflammatory cytokines, tumor necrosis factor-α, interleukin- (IL-) 1β, and IL-6, were decreased when compared with MGO-only exposed HK-2 cells. CTRE alleviated the MGO-induced decrease in nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and antioxidant enzyme mRNA expressions. MGO induced the expression of NADPH oxidase 4 (NOX4); CTRE pretreatment inhibited this induction. Further studies revealed that the NOX4 expression was inhibited owing to the suppression of MGO-induced protein kinase C (PKC) activation following CTRE treatment. Collectively, our data suggest that CTRE attenuates MGO-induced inflammation and oxidative stress via inhibition of PKC activation and NOX4 expression, as well as upregulating the Nrf2-antioxidant enzyme pathway in HK-2 cells.

The effect of total lignans from Fructus Arctii on Streptozotocin-induced diabetic retinopathy in Wistar rats

Ethnopharmacological relevanceFructus Arctii is the dried ripe fruit of Arctium lappa L. (family Asteraceae). It is a well-known Chinese Materia Medica that was included in the Chinese pharmacopoeia because of its traditional therapeutic actions, such as heat removal, detoxification, and elimination of swelling. Since ancient times Fructus Arctii has been used extensively in a number of classical drug formulas to treat type 2 diabetes mellitus. Modern pharmacological studies have shown that certain components of Fructus Arctii have multiple physiological activities on type 2 diabetes and its complications. Aim of the studyWe have reported the inhibitory effect of total lignans from Fructus Arctii (TLFA) on aldose reductase, the key enzyme in the polyol pathway, which is considered to be closely related to the onset of diabetic retinopathy (DR). The present study aimed to observe the preventive and therapeutic effects of TLFA on DR in Streptozotocin (STZ)-induced DR rats. Materials and methodsTLFA was prepared from Fructus Arctii and its content was determined using UV spectrophotometry. The DR model was induced by STZ in Wistar rats. For DR prevention, the animals were gavaged once daily for 9 weeks with TLFA (1.38, 0.69, and 0.35 g/kg/day) as soon as they were confirmed as diabetes models. Pathological changes to retinal tissues and the expression of vascular endothelial growth factor (VEGF) and protein kinase C (PKC) in the retina were detected after TLFA treatment. The effects of TLFA on blood glucose levels and body weight were also observed. For DR treatment, the animals were gavaged once daily for 12 weeks with TLFA (1.38 and 0.69 g/kg/day) at 3 months after they were confirmed as diabetes models. The therapeutic effect was studied using quantitative detection of blood-retina barrier (BRB) breakdown via an Evans Blue leakage assay. ResultsFor DR prevention, after 9 weeks of TLFA administration, histopathological examination of retinal tissue showed that TLFA improved the lesions in the retina. Changes to retinal microstructures such as capillaries, ganglion cells, bipolar cells, and the membrane disk examined by electron microscopy further confirmed that TLFA has a preventive effect on retinopathy. Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) detection showed that TLFA could inhibit retinal cell apoptosis in the diabetic rats, and fasting blood glucose (FBG) levels of rats in the TLFA-treated groups decreased during the experiment. For DR treatment, after 3 months of administration, the amount of dye leakage in the TLFA-administered groups was reduced by more than 50% compared with that in the model group, which indicated that TLFA has a therapeutic effect on middle and late DR. Messenger RNA (mRNA) expression of VEGF and PKCβ2 in the retina detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) showed that TLFA could inhibit the expression of them, which was consistent with the results of immunohistochemistry (IHC). ConclusionTLFA has a preventive and therapeutic effect on DR. Its mechanism of action on DR is related to inhibiting PKC activation and blocking VEGF elevation.

Heterologous regulation of CXCR4 lysosomal trafficking

G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CXC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CXC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficking and degradation. Our results demonstrate that direct activation of PKC via the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal trafficking of CXCR4. In agreement, heterologous activation of PKC by stimulating the chemokine receptor CXCR5 with its ligand, CXCL13, also mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal degradation of CXCR4. Similar to CXCL12, PMA promotes PKC-dependent phosphorylation of serine residues within CXCR4 C-tail that are required for binding and ubiquitination by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 4). However, inhibition of PKC activity does not alter CXCL12-mediated ubiquitination and degradation of CXCR4, suggesting that other kinases are also required. Accordingly, siRNA-mediated depletion of GRK6 results in decreased degradation and ubiquitination of CXCR4. Overall, these results suggest that PKC and GRK6 contribute to unique aspects of CXCR4 phosphorylation and lysosomal degradation to ensure proper signal propagation and termination.

Amelioration of diabetic nephropathy by SGLT2 inhibitors independent of its glucose-lowering effect: A possible role of SGLT2 in mesangial cells

Several clinical studies have shown the beneficial effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on diabetic nephropathy. The underlying mechanisms are not fully understood. We found that administration of canagliflozin at a low dose (0.01 mg/kg/day) did not affect either blood glucose levels or glycosuria, but it improved albuminuria and mesangial expansion in db/db mice to a similar extent as at a high dose (3.0 mg/kg/day) that lowered blood glucose levels. This indicated the existence of a tubular SGLT2-independent reno-protective mechanism. Here we focused on the potential role of SGLT2 in mesangial cells (MCs). Western blot analysis revealed the expression of SGLT2 in cultured mouse MCs. Exposure of MCs to high glucose levels for 72 h significantly increased the expression of SGLT2. Canagliflozin or ipragliflozin (both 100 nM) treatment inhibited glucose consumption in the medium under high-glucose conditions but not under normal-glucose conditions. Furthermore, canagliflozin inhibited high-glucose-induced activation of the protein kinase C (PKC)-NAD(P)H oxidase pathway and increases in reactive oxygen species (ROS) production. Thus, the inhibition of mesangial SGLT2 may cause an inhibition of PKC activation and ROS overproduction in diabetic nephropathy, and this may at least in part account for the reno-protective effect of SGLT2 inhibitors.

Protein Kinase C Inhibition Mediates Neuroblast Enrichment in Mechanical Brain Injuries.

Brain injuries of different etiologies lead to irreversible neuronal loss and persisting neuronal deficits. New therapeutic strategies are emerging to compensate neuronal damage upon brain injury. Some of these strategies focus on enhancing endogenous generation of neurons from neural stem cells (NSCs) to substitute the dying neurons. However, the capacity of the injured brain to produce new neurons is limited, especially in cases of extensive injury. This reduced neurogenesis is a consequence of the effect of signaling molecules released in response to inflammation, which act on intracellular pathways, favoring gliogenesis and preventing recruitment of neuroblasts from neurogenic regions. Protein kinase C (PKC) is a family of intracellular kinases involved in several of these gliogenic signaling pathways. The aim of this study was to analyze the role of PKC isozymes in the generation of neurons from neural progenitor cells (NPCs) in vitro and in vivo in brain injuries. PKC inhibition in vitro, in cultures of NPC isolated from the subventricular zone (SVZ) of postnatal mice, leads differentiation towards a neuronal fate. This effect is not mediated by classical or atypical PKC. On the contrary, this effect is mediated by novel PKCε, which is abundantly expressed in NPC cultures under differentiation conditions. PKCε inhibition by siRNA promotes neuronal differentiation and reduces glial cell differentiation. On the contrary, inhibition of PKCθ exerts a small anti-gliogenic effect and reverts the effect of PKCε inhibition on neuronal differentiation when both siRNAs are used in combination. Interestingly, in cortical brain injuries we have found expression of almost all PKC isozymes found in vitro. Inhibition of PKC activity in this type of injuries leads to neuronal production. In conclusion, these findings show an effect of PKCε in the generation of neurons from NPC in vitro, and they highlight the role of PKC isozymes as targets to produce neurons in brain lesions.

Amelioration of diabetic nephropathy by oral administration of d-α-tocopherol and its mechanisms.

Diabetic nephropathy (DN) is a diabetic vascular complication, and abnormal protein kinase C (PKC) activation from increased diacylglycerol (DG) production in diabetic hyperglycemia is one of the causes of DN. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. In other words, DGK can attenuate PKC activity by reducing the amount of DG. Recently, we reported that intraperitoneally administered d-α-tocopherol (vitamin E, αToc) induces an amelioration of DN in vivo through the activation of DGKα and the prevention of podocyte loss. However, the effect of the oral administration of αToc on DN in mice remains unknown. Here, we evaluated the effect of oral administration of αToc on DN and its molecular mechanism using streptozocin-induced diabetic mice. Consequently, the oral administration of αToc significantly ameliorated the symptoms of DN by preventing the loss of podocytes, and it was revealed that the inhibition of PKCactivity was involved in this amelioration.

Abstract 4059: KPC34: a co-drug that combines a DNA damaging agent with a targeted therapy for the treatment of AML

Abstract AML occurs in the elderly with ~18,000 new US cases in 2014. Mortality is an estimated 10,050 US deaths in 2014. Elderly patient prognosis is a dismal 6.6%, 5 year survival rate. AML treatment involves induction chemotherapy with Ara-C followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Chemoresistance occurs by several mechanisms including reduced activation of Ara-C via down-regulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. The low expression of both dCK and ENT-1 correlates with a poor prognosis in patients with acute leukemias. Recurrent disease is more difficult to treat successfully. Elderly patients often have trouble tolerating repeated chemotherapy treatments. Activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms has been detected in AML. The activity of these isoforms promotes leukemic proliferation and survival by ERK activation and BCL-2. KPC34 is a novel synthetic phospholipid/deoxycytidine analog conjugate. KPC34's novel properties result in improved pharmacokinetics. It overcomes chemoresistance to Ara-C by bypassing ENT-1 uptake and dCK activation. KPC34 is administered orally. It crosses the blood-brain barrier, targeting CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety inhibits PKC activity. The study’s hypothesis is that KPC34 is active against AML in vivo. KPC34 is highly cytotoxic to a variety of leukemic cell lines in vitro, with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). In order to assess the activity of KPC34 in vivo, we injected NSG mice with OCI-AML-3 cells expressing luciferase. Following engraftment, mice were treated with KPC34, Ara-C, or water on days 1, 4 and 7. A significant prolongation of survival was observed in the KCP34-treated mice (median survival 44.5 days vs. 25 days for Ara-C-treated mice, p=0.0033, by log-rank test). Retreatment of the mice with additional KPC34 resulted in a median survival time of 78.5 days (p=0.0028, single KPC34 course vs. 2 additional treatment courses). KPC34 + doxorubicin is superior to the treatment of mice with Ara-C + doxorubicin. In a PDX murine model, KPC 34 showed a reduction in leukemic burden in the liver and spleen. KPC34 is well tolerated in vivo. KPC34 is effective against multiple models of naïve and chemoresistant leukemia. It has two distinct anti-neoplastic mechanisms. Oral administration improves efficacy over i.v. dosing. These data demonstrate KPC34 efficacy in multiple preclinical models of leukemia, and support its continued translation to the clinic. This co-drug has the potential to replace Ara-C-based therapies as a single agent/combo in naïve AML, a single agent in relapsed leukemia, and it reduces the CNS leukemic burden. Citation Format: Gregory L. Kucera, Peter Alexander, Kristin Pladna, Timothy S. Pardee. KPC34: a co-drug that combines a DNA damaging agent with a targeted therapy for the treatment of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2017-4059

Effects of electroacupuncture preconditioning on expression of aquaporin-1 and activity of protein kinase C in myocardium of rats with acute myocardial ischemia-reperfusion injury

To explore the partial action mechanism and the myocardial protective effect differences between electroacupuncture (EA) preconditioning at "Neiguan"(PC 6) and "Taiyuan"(LU 9) in rats with acute myocardial ischemia-reperfusion injury. Ninety-six Wistar rats were randomly assigned into a sham-operation group, a model group, a Neiguan group and a Taiyuan group, 24 rats in each one. The rats in the Neiguan group and Taiyuan group were treated with EA (2 Hz in frequency, 1 mA in intensity) at "Neiguan" (PC 6) and "Taiyuan" (LU 9) respectively, 20 min per treatment, once a day for consecutive 7 days. The rats in the sham-operation group and model group were treated with immobilization for the same time, and no EA was given. The model of myocardial ischemia-reperfusion injury was established in the model group, Neiguan group and Taiyuan group 24 h after the end of EA, while the rats in the sham-operation group were treated with sham operation (no ligation was made during surgery). The myocardial ischemic size, infarction size, activity of protein kinase C (PKC) and expression of aquaporin1 (AQP1) in each group were detected. Compared with sham-operation group, the myocardial ischemic size, infarction size, AQP1 expression and PKC activity in the model group were significantly increased (all P<0.01); compared with the model group and Taiyuan group, the myocardial ischemic size, infarction size, PKC activity and AQP1 expression were significantly decreased in the Neiguan group (P<0.01, P<0.05). By Pearson correlation analysis, the changes of AQP1 expression were positively correlated with those of PKC activity after EA preconditioning. EA preconditioning at "Neiguan" (PC 6) could significantly decrease myocardial AQP1 expression and PKC activity in rats with acute myocardial ischemia-reperfusion injuing, but the effect of EA preconditioning at "Taiyuan"(LU 9) is not obvious; its protective effect is likely to be achieved by inhibiting PKC activity and AQP1 expression.

Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma via ROS, PKC, and Calcium Signaling.

Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 232: 417-425, 2017. © 2016 Wiley Periodicals, Inc.

Madecassoside suppresses proliferation and invasiveness of HGF-induced human hepatocellular carcinoma cells via PKC-cMET-ERK1/2-COX-2-PGE2 pathway

Recent studies showed that Madecassoside (MAD), a pentacyclic triterpene isolated from Centella asitica (L.), was used as a therapeutic agent in wound healing and also as an anti-inflammatory, anti-oxidative activities and anti-aging agent. However, its role in cancer has not been elucidated. In our present study, hepatocyte growth factor (HGF) induced the phosphorylation of its corresponding receptor cMET, increased expression of cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human hepatocellular carcinoma (HCC) cells lines (HepG2 and SMMC-77), and this effect was inhibited by MAD in a dose-dependent manner. In addition, MAD exhibited significant anti-proliferative and anti-invasive effect in HGF-induced HepG2 and SMMC-77 cells. Moreover, MAD inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the protein kinase C (PKC) activity in HGF-induced HepG2 and SMMC-77 cells. This conclusion was consistent with the effect of selective COX-2 inhibitor (NS-398) and knockdown of COX-2 by siRNA on attenuating the proliferation and invasiveness potential, and over-expression of COX-2 on abolishing the effects of MAD on proliferation and invasiveness potential, and was also in parallel with the effect of PKC inhibitor (Bisindolylmaleimide) on inhibiting PKC activity, MEK/ERK1/2 inhibitor (PD98059) inhibited MEK/ERK1/2 pathways in HGF-induced HepG2 and SMMC-77 cells. Collectively, MAD could inhibit the HGF-activated proliferation and invasiveness of HCC cells via regulating the activation of cMET-PKC-ERK1/2-COX-2-PGE2 cascade, which indicated that MAD might help control HGF-linked HCC.

Isoform-specific phosphorylation-dependent regulation of connexin hemichannels.

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.

Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects

Protein kinase C (PKC) plays a key role in neurotransmission in the central nervous system, and targeting PKC domain is considered as a strategy to modulate the anaesthetic effects. In this study, we described a synthetic pipeline to perform high-throughput virtual screening against a large library of 3D structural natural products released recently in order to discover those potential PKC modulators. A total of 100 natural products with top scores were raised, from which 12 promising candidates were tested to determine their inhibitory potencies against PKC. As might be expected, the promiscuous kinase inhibitor staurosporine showed a high PKC inhibitory activity (IC50 = 64 nM), and other two tested compounds, i.e. fisetin and tetrahydropapaverine, were also highly potent with their activities at nanomolar level (IC50 = 370 and 190, respectively).

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin promotes astrocyte activation and the secretion of tumor necrosis factor‐α via PKC/SSeCKS‐dependent mechanisms

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro-inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src-Suppressed-C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)-mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC-induced SSeCKS activation in TCDD-triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD-induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho-p65 expression, and tumor necrosis factor-α secretion in astrocytes. In addition, pre-treatment with PKC inhibitor also attenuated TCDD-induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD-triggered tumor necrosis factor-α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS-dependent mechanisms, highlighting that astrocytes might be important target of TCDD-induced neurotoxicity. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits neurotoxic effects. Here, we show TCDD induces pro-inflammatory responses in astrocytes. TCDD initiates an increase of [Ca2+]i, followed by the activation of PKC, which then induces the activation of Src-suppressed C-kinase substrate (SSeCKS). SSeCKS promotes NF-κB activation and the secretion of TNF-α and nitric oxide in astrocytes.

Overexpression of Diacylglycerol KinaseηEnhances Gαq-Coupled G Protein–Coupled Receptor Signaling

Multiple genome-wide association studies have linked diacylglycerol kinase η (DGKη) to bipolar disorder (BPD). Moreover, DGKη expression is increased in tissue from patients with BPD. How increased levels of this lipid kinase might affect cellular functions is currently unclear. Here, we overexpressed mouse DGKη in human embryonic kidney 293 cells to examine substrate specificity and signaling downstream of endogenous G protein-coupled receptors (GPCRs). We found that DGKη can phosphorylate diacylglycerol (DAG) with different acyl side chains (8:0, 12:0, 18:1). In addition, overexpression of DGKη enhanced calcium mobilization after stimulating muscarinic receptors with carbachol and after stimulating purinergic receptors with ATP. This effect required DGKη catalytic activity, as assessed using a kinase-dead (G389D) mutant and multiple truncation constructs. DGKη was localized throughout the cytosol and did not translocate to the plasma membrane after stimulation with carbachol. Since protein kinase C (PKC) can be activated by DAG and promotes receptor desensitization, we also examined functional interactions between PKC and DGKη. We found that acute activation of PKC with phorbol 12-myristate 13-acetate shortened carbachol-evoked calcium responses and occluded the effect of overexpressed DGKη. Moreover, inhibition of PKC activity with bisindolylmaleimide I (BIM I) produced the same enhancing effect on carbachol-evoked calcium mobilization as overexpressed DGKη, and overexpression of DGKη produced no additional effect on calcium mobilization in the presence of BIM I. Taken together, our data suggest that DGKη enhances GPCR signaling by reducing PKC activation.

Contribution of protein kinase Cα in the stimulation of insulin by the down-regulation of Cavβ subunits

Voltage-gated calcium (Cav) channels and protein kinase C (PKC) isozymes are involved in insulin secretion. In addition, Cavβ, one of the auxiliary subunits of Cav channels, also regulates the secretion of insulin as knockout of Cavβ3 (β3(-/-)) subunits in mice led to efficient glucose homeostasis and increased insulin levels. We examined whether other types of Cavβ subunits also have similar properties. In this regard, we used small interfering RNA (siRNA) of these subunits (20 μg each) to down-regulate them and examined blood glucose, serum insulin and PKC translocation in isolated pancreatic β cells of mice. While the down-regulation of Cavβ2 and β3 subunits increased serum insulin levels and caused efficient glucose homeostasis, the down-regulation of Cavβ1 and β4 subunits failed to affect both these parameters. Examination of PKC isozymes in the pancreatic β-cells of Cavβ2- or β3 siRNA-injected mice showed that three PKC isozymes, viz., PKC α, βII and θ, translocated to the membrane. This suggests that when present, Cavβ2 and β3 subunits inhibited PKC activation. Among these three isozymes, only PKCα siRNA inhibited insulin and increased glucose concentrations. It is possible that the activation of PKCs βII and θ is not sufficient for the release of insulin and PKCα is the mediator of insulin secretion under the control of Cavβ subunits. Since Cavβ subunits are present intracellularly, it is possible that they (1) inhibited the translocation of PKC isozymes to the membrane and (2) decreased the interaction between Cav channels and PKC isozymes and thus the secretion of insulin.

Gestational hypoxia up-regulates protein kinase C and inhibits calcium-activated potassium channels in ovine uterine arteries.

Objective: The present study tested the hypothesis that gestational hypoxia up-regulates protein kinase C (PKC) and inhibits calcium-activated potassium channels (KCa)-mediated relaxations of uterine arteries in pregnancy. Study design: Uterine arteries were isolated from nonpregnant (NPUA) and pregnant (PUA) (~140 day gestation) sheep maintained at either sea level or high altitude (3,820 m for 110 days, PaO2: 60 mmHg). Contractions of uterine arteries were determined. Key findings: In normoxic PUA, selective inhibition of large-conductance KCa (BK) channels significantly enhanced PKC activator phorbol 12, 13-dibutyrate (PDBu)-induced contractions. This effect was abrogated by chronic hypoxia in gestation. Unlike BK channels, inhibition of small-conductance KCa (SK) channels had no significant effect on PDBu-mediated contractions. In normoxic PUA, activation of both BK with NS1619 or SK with NS309 produced concentration-dependent relaxations, which were not altered by the addition of PDBu. However, in uterine arteries treated with chronic hypoxia (10.5% O2 for 48 h), both NS1619- and NS309-induced relaxations were significantly attenuated by PDBu. In NPUAs, inhibition of BK channels significantly enhanced PDBu-induced contractions in both normoxic and hypoxic animals. Conclusion: The results suggest that in the normoxic condition BK inhibits PKC activity and uterine vascular contractility, which is selectively attenuated by chronic hypoxia during gestation. In addition, hypoxia induces PKC-mediated inhibition of BK and SK activities and relaxations of uterine arteries in pregnancy.

Extracellular Chromatin Traps Interconnect Cell Biology, Microbiology, and Immunology

Extracellular Chromatin Traps Interconnect Cell Biology, Microbiology, and Immunology