The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, ß, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

Hepatocyte NLRP3 interacts with PKCε to drive hepatic insulin resistance and steatosis.

Hepatic insulin resistance (IR), as a downstream sequela of nonalcoholic fatty liver disease (NAFLD), is strongly associated with liver steatosis. Despite numerous mechanism advancements, the molecular underpinnings and pathogenesis of hepatic IR, especially regarding the pattern recognition receptors in hepatocytes, remain elusive. Here, we identified hepatocyte NLRP3 as a direct and previously-unresolved driver of hepatic IR to promote steatosis response. Under the model of NAFLD, we identified hepatocyte NLRP3 as a crucial inducer of hepatic IR by undertaking multilayer transcriptomic searches and further confirmed that its expression was increased in the liver tissues from NAFLD patients and mouse models (high-fat diet (HFD), leptin-receptor-deficient (db/db) mice), and in palmitic acid (PA)-induced hepatocytes. Loss- or gain-of-function of hepatocyte-specific NLRP3 in HFD-induced mice ameliorated or exacerbated hepatic IR and steatosis, respectively. Mechanistically, NLRP3 directly bound to and promoted protein kinase C epsilon (PKCε) activation to impair insulin signaling and increase liver steatosis, while inhibition of PKCε activation dampened the beneficial effects seen in HFD-induced NLRP3-deficient mice. Moreover, we performed screening and discovered that the transcription factor Yin Yang 1 (YY1) positively controlled NLRP3 expression. In translational potential, adeno-associated virus serotype 8 (AAV8)-mediated NLRP3 knockdown in the liver alleviated hepatic IR and steatosis in db/db mice, and pharmacological inhibition of NLRP3 markedly alleviated diet-induced metabolic disorders. This finding reveals a previously-unexpected regulatory axis from YY1 to PKCε via NLRP3 induction for metabolic diseases and establishes the YY1-NLRP3-PKCε axis as a potential therapeutic target for NAFLD.

Loss in PKC Epsilon Causes Downregulation of MnSOD and BDNF Expression in Neurons of Alzheimer's Disease Hippocampus.

Oxidative stress and amyloid-ß (Aß) oligomers have been implicated in Alzheimer's disease (AD). The growth and maintenance of neuronal networks are influenced by brain derived neurotrophic factor (BDNF) expression, which is promoted by protein kinase C epsilon (PKCɛ). We investigated the reciprocal interaction among oxidative stress, Aß, and PKCɛ levels and subsequent PKCɛ-dependent MnSOD and BDNF expression in hippocampal pyramidal neurons. Reduced levels of PKCɛ, MnSOD, and BDNF and an increased level of Aß were also found in hippocampal neurons from autopsy-confirmed AD patients. In cultured human primary hippocampal neurons, spherical aggregation of Aß (amylospheroids) decreased PKCɛ and MnSOD. Treatment with t-butyl hydroperoxide (TBHP) increased superoxide, the oxidative DNA/RNA damage marker, 8-OHG, and Aß levels, but reduced PKCɛ, MnSOD, BDNF, and cultured neuron density. These changes were reversed with the PKCɛ activators, bryostatin and DCPLA-ME. PKCɛ knockdown suppressed PKCɛ, MnSOD, and BDNF but increased Aß. In cultured neurons, the increase in reactive oxygen species (ROS) associated with reduced PKCɛ during neurodegeneration was inhibited by the SOD mimetic MnTMPyP and the ROS scavenger NAc, indicating that strong oxidative stress suppresses PKCɛ level. Reduction of PKCɛ and MnSOD was prevented with the PKCɛ activator bryostatin in 5-6-month-old Tg2576 AD transgenic mice. In conclusion, oxidative stress and Aß decrease PKCɛ expression. Reciprocally, a depression of PKCɛ reduces BDNF and MnSOD, resulting in oxidative stress. These changes can be prevented with the PKCɛ-specific activators.

Electroacupuncture Attenuates Induction of Inflammatory Pain by Regulating Opioid and Adenosine Pathways in Mice.

Although inflammatory pain is a common clinical condition, its mechanisms are still unclear. Electroacupuncture (EA), a well-known method of pain management, may reduce inflammatory pain by regulating neurons, astrocytes, and inflammatory signaling pathways. Injections of complete Freund's adjuvant (CFA), which can initiate cell-mediated inflammatory pain, resulted in significant hyperalgesia, which was subsequently prevented by EA. In CFA-injected mice, a dramatic increase was observed in the expression of the following proteins in the dorsal root ganglion and spinal cord dorsal horn: the astrocytic marker GFAP, S100B, RAGE, pPKCε, COX-2, pERK, and pNFκB. These effects were reversed by EA. In addition, mechanical hyperalgesia was significantly reduced in the N6-cyclopentyladenosine (CPA) i.p. or i.m. and endomorphin (EM) i.p. groups. Neither EM i.m. nor EM i.p. exhibited any analgesic effect on thermal hyperalgesia. However, both CPA i.m. and CPA i.p. attenuated thermal hyperalgesia in the mouse inflammatory pain model. We showed that CPA reduced COX-2 and pPKCε expression. However, EM administration did not reduce COX-2 levels. Combined administration of naloxone and rolofylline increased pPKCε and COX-2 pathways. Taken together, our study results revealed a novel and detailed mechanism of EA-induced analgesia that involves the regulation of the opioid and adenosine pathways.

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.

Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cε.

As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCε from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCε and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCε, resulting in preconditioning against cell death induced by OGD/R.

Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.