Role Of PKC Research Articles

Macrophage-conditioned medium enhances tunneling nanotube formation in breast cancer cells via PKC, Src, NF-κB, and p38 MAPK signaling

Tumor-associated macrophages (TAMs) secrete cytokines, chemokines, and growth factors in the tumor microenvironment (TME) to support cancer progression. Higher TAM infiltration in the breast TME is associated with a poor prognosis. Previous studies have demonstrated the role of macrophages in stimulating long-range intercellular bridges referred to as tunneling nanotubes (TNTs) in cancer cells. Intercellular communication between cancer cells via TNTs promotes cancer growth, invasion, metastasis, and therapy resistance. Given the important role of TNTs and macrophages in cancer, the role of macrophage-induced TNTs in chemotherapy drug doxorubicin resistance is not known. Furthermore, the mechanism of macrophage-mediated TNT formation is elusive. In this study, it is shown that the macrophage-conditioned medium (MΦCM) partially mimicked inflammatory TME, induced an EMT phenotype, and increased migration in MCF-7 breast cancer cells. Additionally, secreted proteins in MΦCM induced TNT formation in MCF-7 cells, which led to increased resistance to doxorubicin. Transcriptomic analysis of MΦCM-treated MCF-7 cells showed enrichment of the NF-κB and focal adhesion pathways, as well as upregulation of genes involved in EMT, extracellular remodeling, and actin cytoskeleton reorganization. Interestingly, inhibitors of PKC, Src, NF-κB, and p38 decreased macrophage-induced TNT formation in MCF-7 cells. These results reveal the novel role of PKC and Src in inducing TNT formation in cancer cells and suggest that inhibition of PKC and Src activity may likely contribute to reduced macrophage-breast cancer cell interaction and the potential therapeutic strategy of cancer.

Riluzole, a neuroprotective agent, preserves erectile function following bilateral cavernous nerve injury in male rats.

Neurogenic erectile dysfunction is a highly prevalent complication in men undergoing radical prostatectomy. The underlying mechanisms remain incompletely defined and the effective therapy has been limited. This study aimed to evaluate the protective effect of riluzole and the role of PKC β and excitatory amino acid transporters (EAATs) mediating this effect in a rat model of bilateral cavernous injury (BCNI). A total of 48 male Sprague-Dawley rats were divided into sham, BCNI (at 7, 15 days post-injury) and BCNI treated with riluzole (8 mg/kg/day) groups. Erectile function was measured as maximum intracavernosal pressure (mICP)/mean arterial pressure (MAP) and total ICP/MAP. Changes in protein expressions of phospho (p)-PKC β IIser660 and EAATs were analysed in penis and major pelvic ganglion with western blotting. BCNI decreased erectile function at 7 and 15 days post-injury (mICP/MAP at 4 V: 0.45 ± 0.06 vs 0.84 ± 0.07; 0.34 ± 0.04 vs 0.77 ± 0.04 respectively; p < 0.001) whereas riluzole treatment (for 15 days) preserved erectile function (mICP/MAP at 4 V: 0.62 ± 0.03 vs 0.34 ± 0.04; p < 0.01). The decline in the expression of p-PKC β IIser660 was observed in penis at 7 and 15 days post-injury (p = 0.0003, p = 0.0033), which was prevented by riluzole treatment for 15 days (p = 0.0464). While expressions of EAAT-1 and EAAT-2 decreased in major pelvic ganglion following BCNI (p = 0.0428, p = 0.002), riluzole treatment for 15 days prevented the decrease only in EAAT-2 expression (p = 0.0456). Riluzole improved erectile function via possibly interacting with PKC β II and glutamatergic pathways, as a potential therapeutic candidate for erectile dysfunction.

Deficiency of PKC\u03bb/\u03b9 alleviates the liver pathologic impairment of Schistosoma japonicum infection by thwarting Th2 response

BackgroundThe activation of immune response driven by the eggs of Schistosoma japonicum and the subsequent secretions is the culprit behind granulomatous inflammation and liver fibrosis. Evidence suggests that PKCλ/ι participates in a variety of physiological and pathological processes, including the regulation of metabolism, growth, proliferation and differentiation of cells. However, the role of PKCλ/ι in liver disease caused by Schistosoma japonicum remains unclear.MethodsIn the present study, we observe the pathological changes of egg-induced granulomatous inflammation and fibrosis in the liver of mice infected by Schistosoma japonicum by using conditional PKCλ/ι-knockout mice and wild-type control. Immune cytokines and fibrogenic factors were analyzed by performing flow cytometry and real-time fluorescence quantitative PCR.ResultsThe results of H&E and Masson staining show that the degree of granulomatous lesions and fibrosis in the liver of the infected PKCλ/ι-knockout mice was significantly reduced compared with those of the infected wild-type mice. The mean area of single granuloma and hepatic fibrosis in the PKCλ/ι-knockout mice was significantly lower than that of the wild-type mice (85,295.10 ± 5399.30 μm2 vs. 1,433,702.04 ± 16,294.01 μm2, P < 0.001; 93,778.20 ± 8949.05 μm2 vs. 163,103.01 ± 11,103.20 μm2, P < 0.001), respectively. Serological analysis showed that the ALT content was significantly reduced in the infected knockout mice compared with infected wild-type mice. RT-PCR analysis showed that IL-4 content in knockout mice was significantly increased after Schistosoma japonicum infection, yet the increase was less than that in infected wild-type mice (P < 0.05). PKCλ/ι deficiency led to reduced expression of fibrosis-related factors, including TGF-β1, Col-1, Col-3, α-SMA and liver DAMP factor HMGB1. Flow cytometry analysis showed that the increasing percentage of Th2 cells, which mainly secrete IL-4 cytokines in spleen cells, was significantly lower in PKCλ/ι-deficient mice compared with wild-type mice after infection (P < 0.05).ConclusionsOur data demonstrate that PKCλ/ι deficiency alleviating granulomatous inflammation and fibrosis in the liver of mice with S. japonicum infection by downregulating Th2 immune response is the potential molecular mechanism behind the role of PKCλ/ι in schistosomiasis.Graphical

Impact of deleterious missense PRKCI variants on structural and functional dynamics of protein

Protein kinase C iota (PKCɩ) is a novel protein containing 596 amino acids and is also a member of atypical kinase family. The role of PKCɩ has been explored in neurodegenerative diseases, neuroblastoma, ovarian and pancreatic cancers. Single nucleotide polymorphisms (SNPs) have not been studied in PKCɩ till date. The purpose of the current study is to scrutinize the deleterious missense variants in PKCɩ and determine the effect of these variants on stability and dynamics of the protein. The structure of protein PKCɩ was predicted for the first time and post translational modifications were determined. Genetic variants of PKCɩ were retrieved from ENSEMBL and only missense variants were further analyzed because of its linkage with diseases. The pathogenicity of missense variants, effect on structure and function of protein, association with cancer and conservancy of the protein residues were determined through computational approaches. It is observed that C1 and the pseudo substrate region has the highest number of pathogenic SNPs. Variations in the kinase domain of the protein are predicted to alter overall phosphorylation of the protein. Molecular dynamic simulations predicted noteworthy change in structural and functional dynamics of the protein because of these variants. The study revealed that nine deleterious variants can possibly contribute to malfunctioning of the protein and can be associated with diseases. This can be useful in diagnostics and developing therapeutics for diseases related to these polymorphisms.

PKC-\u03b2 modulates Ca2+ mobilization through Stim1 phosphorylation

BackgroundCalcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool.ObjectivesHere, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1.MethodsSeveral biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope.ResultsOur data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors.ConclusionIn sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

Abstract 13450: Role of PKC in Metabolic Regulation of Endothelial SK Channels

Introduction: Diabetes is associated with increases in metabolites NADH, and PKC. NADH has been recently found to inactivate endothelial SK channels. Overexpressed and activated PKC has been shown to play an important role in diabetes-induced endothelial dysfunction. However, it is undefined if PKC is involved in the NADH dysregulation of endothelial SK channels. Hypothesis: We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels and coronary relaxation function. Methods: SK channel currents of human coronary artery endothelial cells were measured by whole-cell patch-clamp method in the presence or absence of NADH, and/or PKC activator PMA, or PKC inhibitors or endothelial PKC α /PKC β knock-down by using short interfering RNA. The in-vitro coronary arterioles (n = 5) from the harvested atrial tissue samples of patients undergoing cardiac surgery were perfused with NADH in the presence or absence of PKC β inhibitor LY333531. Vascular reactivity was measured by vessel myography in the presence of vasoconstrictor and vasodilators. Results: NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p&lt;0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC β inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p&gt;0.05 vs. control (n=15), Fig. E). PKC α knock-down failed to affect NADH (n=13) and PMA (n=10) inhibition of endothelial SK currents (Fig. F). In contrast, PKC β knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p&gt;0.05, vs. control, Fig. G). Incubation of vessels with NADH significantly reduced relaxation response to the SK activator NS309 and this effect was abolished in the presence of LY333531. Conclusions: The metabolite NADH dysregulation of endothelial SK channels and impairment of endothelium-dependent relaxation function was via the PKC β signaling pathway.

The Role of PKC in Regulating NMDARs in Aluminum-Induced Learning and Memory Impairment in Rats.

Aluminum is a widespread environmental neurotoxicant that can induce Alzheimer's disease (AD)-like damage, such as neuronal injury and impairment of learning and memory. Several studies have shown that aluminum could reduce the synaptic plasticity, but its molecular mechanism remains unclear. In this study, rats were treated with aluminum maltol (Al(mal)3) to establish a toxic animal model and PMA was used to interfere with the expression of PKC. The Morris water maze and open field test were used to investigate the behavioral changes of the rats. Western blotting and RT-PCR were used to detect the expression levels of NMDAR subunits, PKC and CaMKII. The results showed that Al(mal)3 damaged learning and memory function and reduced anxiety in rats. During this process, the expression of PKC was downregulated and it inhibited the expression of NMDARs through the phosphorylation of CaMKII.

A novel transgenic mouse strain expressing PKC\u03b2II demonstrates expansion of B1 and marginal zone B cell populations

Protein kinase Cβ (PKCβ) expressed in mammalian cells as two splice variants, PKCβI and PKCβII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCβII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCβIItg). Our findings demonstrate that homozygous Eµ-PKCβIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCβII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.

Calcium Export from Neurons and Multi-Kinase Signaling Cascades Contribute to Ouabain Neuroprotection in Hyperhomocysteinemia.

Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1–1 nM ouabain on intracellular Ca2+ signaling, mitochondrial inner membrane voltage (φmit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φmit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca2+ overload of neurons and loss of φmit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

PKC\u03b8-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

Pathological retinal neovascularization is the most common cause of vision loss. PKCθ has been shown to play a role in type 2 diabetes, which is linked to retinal neovascularization. Based on these clues, we have studied the role of PKCθ and its downstream target genes JunB and VEGFR3 in retinal neovascularization using global and tissue-specific knockout mouse models along with molecular biological approaches. Here, we show that vascular endothelial growth factor A (VEGFA) induces PKCθ phosphorylation in human retinal microvascular endothelial cells (HRMVECs) and downregulation of its levels attenuates VEGFA-induced HRMVECs migration, sprouting and tube formation. Furthermore, the whole body deletion of PKCθ or EC-specific deletion of its target gene JunB inhibited hypoxia-induced retinal EC proliferation, tip cell formation and neovascularization. VEGFA also induced VEGFR3 expression via JunB downstream to PKCθ in the regulation of HRMVEC migration, sprouting, and tube formation in vitro and OIR-induced retinal EC proliferation, tip cell formation and neovascularization in vivo. In addition, VEGFA-induced VEGFR3 expression requires VEGFR2 activation upstream to PKCθ-JunB axis both in vitro and in vivo. Depletion of VEGFR2 or VEGFR3 levels attenuated VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and retinal neovascularization in vivo and it appears that these events were dependent on STAT3 activation. Furthermore, the observations using soluble VEGFR3 indicate that VEGFR3 mediates its effects on retinal neovascularization in a ligand dependent and independent manner downstream to VEGFR2. Together, these observations suggest that PKCθ-dependent JunB-mediated VEGFR3 expression targeting STAT3 activation is required for VEGFA/VEGFR2-induced retinal neovascularization.

PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells.

Protein kinase C (PKC) is a class of phospholipid-dependent serine/threonine kinases that contribute to cell survival, migration, and invasion. Previous studies demonstrated that PKC participates in insulin secretion. However, the role of PKC in glucose-stimulated insulin secretion (GSIS) remains unclear. Herein, we demonstrated that PKC is an important mediator of insulin secretion and revealed a close relationship between PKC activation and insulin secretion in INS-1E cells. Meanwhile, the presence of PKCα was found to induce TRPC1 phosphorylation in INS-1E cells. TRPC1 phosphorylation levels increased by activating PKCα activity. Inhibition of PKCα activity reduced TRPC1 phosphorylation. Finally, we showed that TRPC1 could reverse the decrease in intracellular Ca2+ levels and reduced insulin secretion induced by treatment with PKCα inhibitor under high glucose conditions. In conclusion, our findings indicated that TRPC1 and PKCα are involved in promoting insulin secretion and that PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells.

The role of PKC and PKD in CXCL12 directed prostate cancer migration

Cancer metastasis is the cause of most cancer related deaths and many cancers are becoming resistant to current therapies. An alternative approach is to investigating signalling pathways that cause cancer cell migration such as chemokine signalling pathways. Such downstream signalling proteins are PKC and PKD. Therefore, we investigated the role of these two proteins in CXCL12 mediated PC3 prostate cancer migration. Whereas PKC and PKD inhibitors do not affect the release of calcium in PC3 prostate cancer cells, both are involved in migration, particularly inhibition of the atypical PKC isoform PKCζ causes the greatest reduction in PC3 cell migration. Classical and/or Novel PKC isoform inhibition changes the shape of the PC3 cells, they show a more rounded morphology, whereas PKD inhibition causes prostate cancer cell to elongate. PKCζ inhibition causes the enlargement of PC3 area possibly due to dysregulated actin cytoskeletal control. These results highlight the importance of verifying which signalling proteins, in which cell and in which chemokine signalling cascade enable cancer cellular migration.

Potentials of PKC in Cancer Progression and Anticancer Drug Development.

PKC is a family of serine-threonine kinases which play crucial roles in the regulation of important signal transduction pathways in mammalian cell-biology. These enzymes are themselves regulated by various molecules that can serve as ligands to the regulatory domains and translocate PKC to membrane for activity. The role of PKC in the modulation of both proliferative and apoptotic signaling in cancer has become a subject of immense interest after it was discovered that PKC regulates a myriad of enzymes and transcription factors involved in carcinogenic signaling. Therefore, PKC has served as an attractive target for the development of newer generation of anti-cancer drugs. The following review discusses the potential of PKC to be regarded as a target for anti-cancer therapy. We also review all the molecules that have been discovered so far to be regulators/activators/inhibitors of PKC and also how far these molecules can be considered as potential candidates for anti-cancer drug development based on PKC.

1787-P: Protein Kinase C (PKC) Negatively Regulate Neuronal Insulin Signaling In Vitro

PKCs are serine/threonine kinases sub-divided into three categories, namely, conventional, novel and atypical. Depending upon the isoform some have been reported to regulate insulin signaling positively and some negatively, in peripheral insulin responsive tissues like, skeletal muscles, adipocytes and hepatocytes through PI3K-Akt pathway, leading to glucose uptake. Though, peripheral tissues have been extensively studied, neuronal insulin signaling is less explored. Neuronal insulin signaling is required for maintaining cognitive abilities including synaptic plasticity and memory formation. Therefore, it is important to explore the role of PKC in neuronal insulin signaling. The objective of the present study was to elucidate the role of PKC in regulating insulin signaling in neuronal cells in vitro. To study that, differentiated Neuro-2a (N2a) cells were cultured in vitro and treated with or without phorbol 12-myristate 13-acetate (PMA), a well-known activator of PKC, with or without insulin stimulation and the effect was tested on the insulin signaling proteins. PKC when activated by PMA (400nM) and stimulated with insulin (100nM), down-regulated the activity of Akt by 25% (serine-473) and 20% (threonine-308). Testing of further downstream insulin signaling molecules under the same conditions demonstrated decrease in activity of AS160 by 20% and GSK3β by 20%. However, there was no change in the activity of GSK3α, suggesting a specificity of PKC towards GSK3β in neurons. To determine the functionality of these phosphorylations/activations, glucose uptake measurement showed that PKC decreased glucose uptake by 25%. Taken together these data strongly demonstrates for the first time that PKC negatively regulates insulin signaling cascade via Akt-AS160-GSK3β-glucose uptake pathway in neuronal cells. Further knowledge in this regard will pave the way for future understanding of insulin signaling in neuronal system and possibly neurodegenerative disorders. Disclosure D. Mishra: None. C.S. Dey: None. Funding Council of Scientific and Industrial Research, Government of India, New Delhi; Department of Science and Technology (SR/SO/HS-238/2012, SR/S2/JCB-24/2008(G))

Role of PKC? in Bladder Cancer Progression

Role of PKC? in Bladder Cancer Progression

Abstract 464: Cell death and senescence: Role of protein kinase C in promoting senescence

Abstract Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells undergo massive genome modulation including induction of the senescence-associated secretory phenotype (SAPS). Components of SASP can implement cell cycle arrest or recruit the immune system to clear senescent cells, thus contributing to tumor suppression. However, some secreted pro-inflammation molecules, act as pro-tumorigenic agents contributing to tumor progression, suggesting that elimination of senescent cells could be beneficial. Very few studies showed a role for PKC in senescence. Here we show that PKCeta, an epithelial specific and anti-apoptotic kinase (epithelia is the origin of about 90% of human tumors), promotes senescence induced by oxidative stress and DNA damage. Using PKCeta-knockdown breast adenocarcinoma MCF-7 cells we show that PKCeta promotes senescence induced by oxidative stress and DNA damage via its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and to modulate transcription of major components of SASP such as IL-6 and IL-8. Moreover, we demonstrate that PKCeta creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor. Thus, the expression of PKCeta modulates major components of SASP. Furthermore, our studies demonstrate that PKCeta interferes with γH2AX phosphorylation, which mark DNA double-strand breaks for repair. The phosphorylation of γH2AX may reflect the extent of DNA breaks but also the cellular response to DNA damage. Our experiments suggest that the response to DNA damage (repair processes) was more efficient in PKCeta-knockdown cells. The phosphorylation on ATM and Chk2 was also lower in PKCeta expressing cells. Revealing the molecular regulators of senescence will improve our ability to develop new therapeutic strategies for clearing tumor cells. Our studies showing a role for PKCeta in senescence could identify PKCeta and/or its activated signaling cascades as targets for intervention senescence induction. Since PKCeta is highly expressed in epithelia that are the origin of about 90% of human tumors, this may provide critical inhibitors as therapeutic adjuvants for different types of carcinomas. Citation Format: Etta Livneh, Assaf Ben Ari, Udi Zurgil. Cell death and senescence: Role of protein kinase C in promoting senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 464.

PKC Modulates sFlt1 Production by Human Placental Trophoblasts in Response to Oxidized LDL

Preeclampsia (PE) is a leading cause of maternal and perinatal mortality and mortality, and its prevalence is 4 fold higher in women with diabetes than those without: the underlying mechanism is unclear. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt1) plays a key role in PE pathogenesis. Evidence suggests that PKC activation, which is associated with the development of diabetic vascular complications, may mediate the enhanced sFlt1 release in non-trophoblast cells. We aimed to determine the role of PKC in the regulation of sFlt1 expression in placental trophoblasts, and whether diabetic conditions upregulate sFlt1 expression via the PKC pathway. Human trophoblasts HTR8/SVneo were treated in three separate experiments with PKC activator phorbol-12-myristate-13-acetate (PMA), or diabetes stimuli ’heavily oxidized, glycated’ low-density lipoproteins (HOG-LDL) vs. native LDL ± high glucose (30 mM), over 24h, with or without PKC inhibitor GF109203X (GX). Both sFlt1 mRNA expression (RT-PCR) and protein release (ELISA) were measured. Compared to the control, PMA (5 nM) increased sFlt1 mRNA expression (2.5 fold, p&amp;lt;0.001) and protein release (383.8 ± 27.4 vs. 118.3 ± 21.3 pg/ml, p&amp;lt;0.001) in trophoblasts; these effects were entirely abrogated by the pre-treatment with 5 uM GX for mRNA expression (by 54%, p&amp;lt;0.001) and protein release (115.5 ± 20.9 pg/ml, p&amp;lt;0.001) vs. PMA-treated cells. Similarly, HOG-LDL (50 µg/ml) increased sFlt1 mRNA expression (3.7 fold, p&amp;lt;0.001) and protein release (126.4 ± 16.3 vs. 55.1 ± 2.4 pg/ml, p&amp;lt;0.001), and the both effects were partially abolished by GX (by 33-37%, p&amp;lt;0.05). High glucose did not significantly affect sFlt1 expression, or alter the effect of HOG-LDL. The data suggest that modified lipoproteins may promote PE development in women with diabetes, at least in part via PKC-mediated upregulation and release of sFlt1. These findings provide new insights into disease mechanisms, and may lead to new targets for prevention and treatment of PE. Disclosure R.P. Chow: None. J. Zhao: None. J. Yu: None.

Ischemia/Reperfusion-Induced Translocation of PKC\u03b2II to Mitochondria as an Important Mediator of a Protective Signaling Mechanism in an Ischemia-Resistant Region of the Hippocampus

Emerging reports indicate that activated PKC isoforms that translocate to the mitochondria are pro- or anti-apoptotic to mitochondrial function. Here, we concentrate on the role of PKCβ translocated to mitochondria in relation to the fate of neurons following cerebral ischemia. As we have demonstrated previously ischemia/reperfusion injury (I/R) results in translocation of PKCβ from cytoplasm to mitochondria, but only in ischemia-resistant regions of the hippocampus (CA2-4, DG), we hypothesize that this translocation may be a mediator of a protective signaling mechanism in this region. We have therefore sought to demonstrate a possible relationship between PKCβII translocation and ischemic resistance of CA2-4, DG. Here, we reveal that I/R injury induces a marked elevation of PKCβII protein levels, and consequent enzymatic activity, in CA2-4, DG in the mitochondrial fraction. Moreover, the administration of an isozyme-selective PKCβII inhibitor showed inhibition of I/R-induced translocation of PKCβII to the mitochondria and an increase in neuronal death following I/R injury in CA1 and CA2-4, DG in both an in vivo and an in vitro model of ischemia. The present results suggest that PKCβII translocated to mitochondria is involved in providing ischemic resistance of CA2-4, DG. However, the exact mechanisms by which PKCβII-mediated neuroprotection is achieved are in need of further elucidation.

Enhancement of spinal dorsal horn neuron NMDA receptor phosphorylation as the mechanism of remifentanil induced hyperalgesia: Roles of PKC and CaMKII.

BackgroundModulation of N-methyl-D-aspartate receptor subunits NR1 and NR2 through phosphorylation mediates opioid-induced hyperalgesia, and activations of protein kinase C and extracellular signal-regulated kinase 1/2 potentiate while activation of calcium/calmodulin-dependent protein kinase II inhibits opioid-induced hyperalgesia. However, the mechanism of opioid-induced hyperalgesia development and in particular the potential interplay between N-methyl-D-aspartate receptors and protein kinase C or calcium/calmodulin-dependent protein kinase II or extracellular signal-regulated kinase 1/2 in the development of remifentanil-induced hyperalgesia is unclear.MethodsRemifentanil (1 µg ċ kg−1 ċ min−1) was given intravenously over 60 min in rats, followed by the infusion of either vehicle solution or the respective inhibitors of protein kinase C (chelerythrine), extracellular signal-regulated kinase II (KN93), or extracellular signal-regulated kinase 1/2 (PD98059). Thereafter, the pain behaviors were evaluated by the paw withdrawal mechanical threshold and paw withdrawal thermal latency. In in vitro studies, fetal spinal cord dorsal horn neurons were primary cultured in the presence of 4 nM remifentanil for 60 min, and then the remifentanil was washed out and replaced immediately by culturing in the absence or presence of chelerythrine, KN93 or PD98059, respectively for up to 8 h. The expressions of N-methyl-D-aspartate receptors subunits and their phosphorylation (NR1, NR2B, p-NR1, p-NR2B) were analyzed by Western blotting after the completion of treatments. Functional changes of N-methyl-D-aspartate receptors were evaluated by electrophysiologic recordings of N-methyl-D-aspartate currents.ResultsRemifentanil induced significant thermal and mechanical hyperalgesia, which were significantly attenuated by Chelerythrine or KN93 but not PD98059. The expressions of NR1, NR2B, p-NR1, and p-NR2B were increased significantly and progressively over time after remifentanil administration, and these increases were all significantly attenuated by either chelerythrine or KN93 but not PD98059. Intriguingly, N-methyl-D-aspartate receptor functional enhancement induced by remifentanil was attenuated by Chelerythrine, KN93, and PD98059.ConclusionsIt is concluded that the enhancements in function and quantity of N-methyl-D-aspartate receptor via phosphorylation of its subunits through protein kinase C and calcium/calmodulin-dependent protein kinase II activation may represent the major mechanism whereby remifentanil induced hyperalgesia.