Down-regulation Of Protein Kinase C Research Articles

The rapid activation of cPKCβII by progesterone results in the negative regulation of Ca 2+ influx in human resting T cells.

Progesterone-stimulated rapid suppression of phytohemagglutinin (PHA)-activated sustained membrane Ca 2+ influx is revealed by Mn 2+ quenching fura-2 fluorescence. Ca 2+ influx suppression results in immunosuppression of T-cell proliferation. Downregulation of protein kinase C (PKC) activity by phorbol 12-myristate 13-acetate (PMA) enhances the PHA-activated increase in sustained intracellular Ca 2+ concentration ([Ca 2+ ] i ) via Ca 2+ influx in T cells. Conventional PKC (cPKC) inhibitors also enhance the [Ca 2+ ] i increase in resting T cells caused by progesterone. This study explores whether cPKC activation by progesterone results in suppression of Ca 2+ influx in resting T cells. Progesterone, its analogs (R5020/Org OD 02-0), and plasma membrane-impermeable progesterone-bovine serum albumin conjugate were used to stimulate human resting T cells. Inhibitors and PKC downregulation by PMA were used to investigate whether cPKC affects Ca 2+ influx. Progesterone and analogs dose-dependently suppressed Ca 2+ influx in T cells. One cPKC inhibitor, Ro318220, attenuated Ca 2+ influx suppression, and enhanced the increase in [Ca 2+ ] i caused by progesterone and analogs. U73122 did not affect Ca 2+ influx suppression but did decrease the [Ca 2+ ] i increase. Ca 2+ influx suppression was not attenuated by the cPKCα/βI isoform-selective inhibitor, Go6976, nevertheless, a cPKCβI/βII isoform-selective inhibitor, LY333531 did. Ca 2+ influx suppression was attenuated by the cPKCβII-specific inhibitor CGP53353. After PKC downregulated by PMA, Ca 2+ influx suppression by progesterone and analogs was almost abolished in parallel with a massive reduction in cPKCβII expression. This suggests cPKCβII activation by progesterone and analogs mediate Ca 2+ influx suppression in resting T cells. Nongenomic membrane activation of cPKCβII by progesterone causes immunosuppression via negative regulation of Ca 2+ influx into human resting T cells. This prevents resting T-cell activation and proliferation, which protects the fetus from maternal immune attack while decreasing maternal autoimmune disease flare-ups during pregnancy. Thus, cPKCβII modulators might provide a new therapeutic approach to balancing T-cell tolerance and immunity.

Pingchong Jiangni recipe through nerve growth factor/transient receptor potential vanilloid 1 signaling pathway to relieve pain in endometriosis model rats

Ethnopharmacological relevancePingchong Jiangni recipe (PJR) is often used in the treatment of endometriosis (EM). This formula has been clinically validated by the State Administration of Traditional Chinese Medicine Key Specialties Collaborative Group for its therapeutic efficacy. Recently, our research team also confirmed that PJR has a shrinking effect on ovarian chocolate cysts. Additionally, PJR was also found to have a shrinking effect on EM lesions; however, the mechanism by which this effect occurs remains unclear. Aim of the studyTo explore the mechanisms by which PJR relieves pain in patients with EM. Materials and methodsA rat model of EM was established by autologous transplantation. PJR (3.78 g/kg, 7.56 g/kg, and 15.12 g/kg) was orally administered for 21 days. The rat grimace scale (RGS) score and paw withdrawal threshold (PWT) were measured at a fixed time during the experiment. Hematoxylin and eosin staining was performed to observe histopathological changes in EM rats after administration, enzyme-linked immunosorbent assay to evaluate the plasma expression of tumor necrosis factor-α (TNF-α) and nerve growth factor (NGF), and immunohistochemistry and western blotting to identify differences in the expression of pain-related factors in EM rats. ResultsThe medium-dose group of PJR (7.56 g/kg) had the best effect on relieving pain in EM rats by reducing RGS, increasing PWT, reducing the ectopic endometrium, improving the cellular structure of the lesion, and reducing TNF-α and NGF levels. However, PJR significantly decreased the expression of transient receptor potential vanilloid 1 (TRPV1), phosphorylated TRPV1 (p-TRPV1), protein kinase C (PKC), and NGF. ConclusionThe mechanism by which PJR relieves EM pain may be through the downregulation of NGF, PKC, and TRPV1 expression.

Thrombin Stimulation of Human Endothelial Cell Phospholipase D Activity. Regulation by Phospholipase C, Protein Kinase C, and Cyclic Adenosine 3' 5’ -Monophosphate

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

ABSTRACTOver the last years extensive kinase-mediated regulation of a number of voltage-gated potassium (Kv) channels important in cardiac electrophysiology has been reported. This includes regulation of Kv1.5, Kv7.1 and Kv11.1 cell surface expression, where the kinase-mediated regulation appears to center around the ubiquitin ligase Nedd4-2. In the present study we examined whether Kv1.4, constituting the cardiac Ito,s current, is subject to similar regulation. In the epithelial Madin-Darby Canine Kidney (MDCK) cell line, which constitutes a highly reproducible model system for addressing membrane targeting, we find, by confocal microscopy, that Kv1.4 cell surface expression is downregulated by activation of protein kinase C (PKC) and AMP-activated protein kinase (AMPK). In contrast, manipulating the activities of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serum and glucocorticoid-regulated kinase 1 (SGK1) were without effect on channel localization. The PKC and AMPK-mediated downregulation of Kv1.4 membrane surface localization was confirmed by two-electrode voltage clamp in Xenopus laevis oocytes, where pharmacological activation of PKC and AMPK reduced Kv1.4 current levels. We further demonstrate that unlike related Kv channels, Kv1.4 current levels in Xenopus laevis oocytes are not reduced by co-expression of Nedd4-2, or the related Nedd4-1 ubiquitin ligase. In conclusion, we demonstrate that the surface expression of Kv1.4 is downregulated by the two kinases AMPK and PKC, but is unaffected by PI3K-SGK1 signaling, as well as Nedd4-1/Nedd4-2 activity. In the light of previous reports, our results demonstrate an impressive heterogeneity in the molecular pathways controlling the surface expression of highly related potassium channel subunits.

Protein kinase C downregulation induces senescence via FoxO3a inhibition in HCT116 and HEK293 cells

We investigated the impact of protein kinase C (PKC) on cellular senescence. The PKC activity and expression of conventional PKC (cPKC) and atypical PKC (aPKC) isoforms decreased during replicative senescence in IMR-90 cells. Forced inhibition of cPKC or aPKC induced the activation of senescence markers, including senescence-associated β-galactosidase activity and reactive oxygen species (ROS)-p53-p21Cip1/WAF1 axis in HCT116 and HEK293 cells. PKC inhibition triggered the nuclear exportation of FoxO3a via stimulation of AKT-mediated phosphorylation of FoxO3a, and thereby decreased the transcription of FoxO3a target genes. Conversely, ectopic expression of the PKC isoforms led to stimulation of the nuclear import of FoxO3a and expression of the FoxO3a target genes. Ectopic FoxO3a expression attenuated ROS accumulation and senescent phenotypes induced by PKC inhibition. Therefore, this study suggests for the first time that downregulation of PKC induces senescence through the AKT-FoxO3a-ROS-p53-p21Cip1/WAF1 pathway in HCT116 and HEK293 cells.

ErbB4 cleavage by gonadotropin-releasing hormone receptor stimulation in cultured gonadotroph cells

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G-protein-coupled receptors, and its stimulation activates extracellular signal-regulated protein kinase (ERK). In the present study, we first examined the actions of GnRH on the ErbB family using two types of cultured gonadotroph cells. As reported previously, AG1478, an inhibitor of the ErbB family tyrosine kinase, inhibited GnRH-induced ERK activation in undifferentiated gonadotroph αT3-1 cells. However, AG1478 did not inhibit ERK activation in differentiated gonadotroph LβT2 cells, suggesting that transactivation of the ErbB family was not necessary for ERK activation in LβT2 cells. We found that ErbB4 was expressed in αT3-1 cells but not in LβT2 cells. GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment in αT3-1 cells. Pharmacological experiments suggested that Gq/11 and tumor necrosis factor-α-converting enzyme (TACE) were essential for GnRH-induced ErbB4 cleavage. GnRH increased the phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), indicating that GnRH activated protein kinase C (PKC). Down-regulation of PKC and bisindolylmaleimide I, a PKC inhibitor, strongly inhibited the GnRH-induced cleavage of ErbB4. It was surprising that GnRH treatment of LβT2 cells after overexpression of ErbB4 induced ErbB4 cleavage in a TACE-dependent manner. ErbB4 cleavage was induced also by treatment of αT3-1 cells, ErbB4-overexpressing LβT2 cells, and immortalized GnRH neurons (GT1-7 cells) with leuprorelin acetate. These results may suggest that the pharmacological effects of leuprorelin acetate are conducted through TACE-mediated proteolysis of membrane proteins, including ErbB4, in gonadotroph cells and GnRH neurons.

Epigenetic Mechanisms: New Targets for Heart Failure Pharmacopuncture.

Pharmacopuncture treatments are proposed for cardiovascular disease and heart failure [1]. Heart failure results from complex environment-gene interactions. β-adrenergic receptors and protein kinase A (PKA) interact with histones and related segments of deoxyribonucleic acid (DNA) at promoter regions of genes for messenger ribo nucleic acid (mRNA) transcription. This increases cardiac muscle mass and contractility in normal hearts. In heart failure, activation of β-adrenergic receptors and PKA promote pathological hypertrophy and decreased contractility. Experimental models show that prenatal exposure to hypoxia, cocaine, or nicotine increases susceptibility to heart failure when animals reach adulthood. Hypermethylation of DNA is an epigenetic mechanism associated with downregulation of the protein kinase C (PKC) gene in such models of heart failure. Downregulation of PKC is also produced by the stress-related hormone norepinephrine with upregulation of the hypoxia-inducible differentiation regulator Nix in norepinephrine-induced cardiac fibrosis [2]. Norepinephrine is also the main mediator of sympathetic neural activity. Sympathetic neural overactivity, a significant cofactor in human heart failure, is, therefore, implicated as a cofactor in this epigenetic mechanism for heart failure. Other epigenetic mechanism for cardiac hypertrophy and heart failure involve endothelin- 1 induced downregulation of the cardiac myocyte differentiation factor RE1-silencing transcription signaler (REST) and GATA zinc-finger domain-containing protein-1 (GATAD1) induced inhibition of histone deacetylase (HDAC-2) in cardiac myocytes harvested from autosomal-recessive dilated cardiac myopathy patients with heart failure [3]. In contrast, both the HDAC-2 in cardiac myocytes and the HDAC-1 in cardiac fibroblasts are upregulated in experimental animal models of congestive heart failure [4]. A prominent role for inflammation in heart failure is suggested by tumor necrosis factor (TNF-α), a proinflammatory cytokine, inducing hypermethylation of the sarcoplasmic reticulum calcium ATPase (SERCA-2A) gene in cardiac myocytes, associated with diastolic dysfunction and heart failure. An important role for epigenetic mechanisms in heart failure is also suggested by HDAC-dependent stimulation of the stress-apoptosis intracellular signaling pathway, which induces hypertrophy of both cardiac and vascular smooth muscle [5].

Anticarcinogenic action of quercetin by downregulation of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) via induction of p53 in hepatocellular carcinoma (HepG2) cell line.

Protein kinase C (PKC) is a key regulator of cell growth and differentiation in mammalian cells and hyperactivation of PKC is believed to play an important role in tumor progression. PKC is downstream to signaling protein of phosphatidylinositol 3-Kinase (PI3K), a known up-regulator of cell proliferation and survival. Accumulation of reactive oxygen species (ROS) triggers oxidative stress in the tumor microenvironment, leading to the hyperactivation of various oxidative stress-stimulated signaling molecules. Quercetin (QUE) is a naturally occurring dietary flavonoid having antioxidant properties. QUE is reported to show antitumor activity both in vitro and in vivo; however, the molecular mechanism is yet to be thoroughly explored. HepG2 cells display cellular functions similar to the normal hepatocytes with high degree of morphological and functional differentiation, therefore HepG2 cell line is chosen as the suitable model for drug targeting. Present study is aimed to establish the signaling pathway involved in the anticarcinogenic action of QUE in HepG2 cell line. HepG2 cells were treated with different doses of QUE. Protein level and gene expression were analysed by Western blotting and RT-PCR, respectively. PKC activity was measured by non-radioactive-tagged phosphorylation. Results showed downregulation of expression of PI3K, PKC, COX-2 and ROS caused by QUE. Additionally, QUE enhanced the expression of p53 and BAX in HepG2 cells. Overall, results of the current study suggested that QUE elicited anticarcinogenic action by upregulation of p53 and BAX in HepG2 cells via downregulation of ROS, PKC, PI3K and COX-2, confirming our earlier report on the animal model.

The C-Terminal V5 Domain of Protein Kinase Cα is a Multi-Functional Intrinsically Disordered Protein Module

Protein Kinase C (PKC) family of isozymes regulate a multitude of signaling pathways that control cell growth, differentiation, and apoptosis. The extreme C-terminal V5 domain has been identified as a key player in maturation, activation and down-regulation of PKCs, but the molecular basis of these events remains poorly understood. We report the first large-scale purification and NMR characterization of the V5 domain from PKCα (V5α) and its phosphorylation-mimicking variant. The combined analysis of NMR chemical shifts and circular dichroism data revealed that both V5α constructs are intrinsically disordered protein domains. Unexpectedly, we found that V5α has a propensity to partition into membrane mimetics acquiring a partial helical structure. Our data suggest that V5α anchors its parent enzyme to membranes during the maturation process. Using NMR techniques, we obtained direct evidence that V5α interacts with another domain of PKCα -- the C2 regulatory domain. This interaction is mediated by the phosphorylated hydrophobic motif of V5α and is enhanced by Ca2+. Similarly, the affinity of C2 to Ca2+ is enhanced in the presence of the phosphorylated hydrophobic motif. These findings indicate that V5α may function as an intra-molecular protein interaction module that sensitizes PKCα to Ca2+ ions. The third aspect of V5α function pertains to its interactions with Pin1, a peptidyl-prolyl isomerase that has been implicated in the down-regulation of conventional PKCs. Pin1 catalyzes proline isomerization of the phosphorylated Ser/Thr-Pro motif. Our preliminary NMR data demonstrate that Pin1 interacts with the hydrophobic motif of V5α, which is a non-canonical site that lacks a proline after the phosphorylated serine. Our data support the hypothesis that V5α serves as a phosphorylation-dependent docking site for Pin1. Acknowledgments: This work was supported by the Welch Foundation (A-1784).

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P1) phosphorylation was studied. Activation of S1P1 receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P1 phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P1 phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P1 phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P1 immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P1 phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P1.

In Vitro Biomechanical Strain Regulation of Fibroblast Wound Healing

Strain-directed therapy such as vacuum compression and manual manipulative therapies are clinically effective, but their cellular and molecular mechanisms are not well understood. To determine the effects of modeled myofascial release (MFR) on fibroblast wound healing and to investigate the potential role of nitric oxide (NO) in mediating these responses. Using an in vitro scratch wound strain model, the authors investigated human fibroblast wound healing characteristics in response to injurious repetitive motion strain (RMS) and MFR. Secretion of NO was induced with interleukin-1β and sodium nitroprusside and inhibited with NO synthase inhibitor L-N(G)-monomethyl arginine citrate (L-NMMA) to determine the effects of NO on wound healing. Protein microarray was also performed to evaluate the expression of intracellular protein and activation of protein kinase G (PKG), extracellular signal-regulated kinase (ERK1/2), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), the downstream effectors in the NO pathway. Fibroblasts that received RMS resulted in reduced wound closure rates (vs nonstrain, P<.05), which are partially attenuated by a single dose of MFR. Interleukin-1β and exogenous NO did not appear to have an effect on nonstrained fibroblast wound healing. However, strained fibroblasts appeared to express increased sensitivity to NO. The authors also observed a 12.2% increase in NO secretion, an increase in PKG activation, and a downregulation of PKC and PI3K inhibitory domain in the combined strain group. If clinically translatable, these data suggest that mechanical strain such as vacuum compression therapy and manual manipulative therapy may modify PKC and PI3K to sensitize fibroblasts to NO and improve wound healing by promoting cell proliferation and migration by means of PKC and PKG signaling.

PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts

Primary cells respond to irradiation by activation of the DNA damage response and cell cycle arrest, which eventually leads to senescence or apoptosis. It is not clear in detail which signaling pathways or networks regulate the induction of either apoptosis or senescence. Primary human fibroblasts are able to withstand high doses of irradiation and to prevent irradiation-induced apoptosis. However, the underlying regulatory basis for this phenotype is not well understood. Here, a kinetic network analysis based on reverse phase protein arrays (RPPAs) in combination with extensive western blot and cell culture analyses was employed to decipher the cytoplasmic and nuclear signaling networks and to identify possible antiapoptotic pathways. This analysis identified activation of known DNA damage response pathways (e.g., phosphorylation of MKK3/6, p38, MK2, Hsp27, p53 and Chk1) as well as of prosurvival (e.g., MEK-ERK, cAMP response element-binding protein (CREB), protein kinase C (PKC)) and antiapoptotic markers (e.g., Bad, Bcl-2). Interestingly, PKC family members were activated early upon irradiation, suggesting a regulatory function in the ionizing radiation (IR) response of these cells. Inhibition or downregulation of PKC in primary human fibroblasts caused IR-dependent downregulation of the identified prosurvival (CREB phosphorylation) and antiapoptotic (Bad phosphorylation, Bcl-2) markers and thus lead to a proliferation stop and to apoptosis. Taken together, our analysis suggests that cytoplasmic PKC signaling conditions IR-stressed MRC-5 and IMR-90 cells to prevent irradiation-induced apoptosis. These findings contribute to the understanding of the cellular and nuclear IR response and may thus eventually improve the efficacy of radiotherapy and help overcome tumor radioresistance.

Role of protein kinase C in the activation of store-operated Ca2+ entry in airway smooth muscle cells

Store-operated Ca(2+) channels (SOCs) are plasma membrane Ca(2+) permeable channels activated by depletion of intracellular Ca(2+) store. Ca(2+) entry through SOCs is known as store-operated Ca(2+) entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca(2+) fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ɛ isoforms in rat ASMCs. PKCα-selective inhibitor Gö6976 and PKCɛ-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid β specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.

Chronic social defeat up-regulates expression of norepinephrine transporter in rat brains

Stress has been reported to activate the locus coeruleus (LC)–noradrenergic system. However, the molecular link between chronic stress and noradrenergic neurons remains to be elucidated. In the present study adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD) for 4 weeks. Measurements by in situ hybridization and Western blotting showed that CSD significantly increased mRNA and protein levels of the norepinephrine transporter (NET) in the LC region and NET protein levels in the hippocampus, frontal cortex and amygdala. CSD-induced increases in NET expression were abolished by adrenalectomy or treatment with corticosteroid receptor antagonists, suggesting the involvement of corticosterone and corticosteroid receptors in this upregulation. Furthermore, protein levels of protein kinase A (PKA), protein kinase C (PKC), and phosphorylated cAMP-response element binding (pCREB) protein were significantly reduced in the LC and its terminal regions by the CSD paradigm. Similarly, these reduced protein levels caused by CSD were prevented by adrenalectomy. However, effects of corticosteroid receptor antagonists on CSD-induced down-regulation of PKA, PKC, and pCREB proteins were not consistent. While mifeprestone and spironolactone, either alone or in combination, totally abrogate CSD effects on these protein levels of PKA, PKC and pCREB in the LC and those in the hippocampus, frontal cortex and amygdala, their effects on PKA and PKC in the hippocampus, frontal cortex and amygdala were region-dependent. The present findings indicate a correlation between chronic stress and activation of the noradrenergic system. This correlation and CSD-induced alteration in signal transduction molecules may account for their critical effects on the development of symptoms of major depression.

Inhibition of protein kinase C promotes differentiation of neuroblastoma × glioma NG108-15 hybrid cells

Differentiation of neuroblastoma × glioma NG108-15 hybrid cells can be induced by different means, but the mechanisms involved are unclear. Our aim was to characterize the role of protein kinase C (PKC) in this process. The PKCs present in NG108-15 cells, i.e. PKCα, PKCδ, PKCε and PKCζ, were inhibited using a cocktail of Go6983 and Ro318220 or were downregulated by treatment with phorbol 12-myristate 13-acetate (PMA). In high-glucose Dulbecco's modified Eagle medium, neuritogenesis was induced by 24 h treatment with a cocktail of Go6983 and Ro318220 or by 48 h treatment with PMA, the latter process thus requiring a longer treatment. However, when cells treated with PMA for only 24 h were placed in extracellular standard salts solution, e.g. Locke's buffer, for 3 h, morphological and functional differentiation occurred, with rounding of the cell body, actin polymerization subjacent to the plasma membrane and an increase in voltage-sensitive Ca(2+) channel activity in the absence of cell death. This rapid differentiation was not due to autophagy, growth arrest or increased cyclic AMP response element binding protein phosphorylation, but coincided with combined activation of p38 mitogen-activated protein kinase (MAPK) and inhibition of extracellular signal-regulated kinase (ERK) and Akt, as confirmed by the effects of selective inhibitors. Furthermore, PKC activation blocked thapsigargin-induced neuritogenesis, whereas PKC downregulation did not. These results show that PKC downregulation promotes differentiation and this effect is accelerated by exposure to Locke's buffer. Although this experimental paradigm cannot be related to the in vivo situation and disease, it implies that combined inhibition of Akt and p44/p42 ERK and activation of p38 MAPK promotes differentiation.

Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.

1950 PROTEIN KINASE C INHIBITS SPONTANEOUS AND NON-VOIDING CONTRACTIONS IN DETRUSOR SMOOTH MUSCLE: ROLE AND INTERACTION WITH BK CHANNELS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Non-Neurogenic Voiding Dysfunction1 Apr 20111950 PROTEIN KINASE C INHIBITS SPONTANEOUS AND NON-VOIDING CONTRACTIONS IN DETRUSOR SMOOTH MUSCLE: ROLE AND INTERACTION WITH BK CHANNELS Joseph Hypolite, Qi Lei, Shaohua Chang, Anna Malykhina, Stephen Zderic, Alan Wein, Stephan Butler, and Samuel Chacko Joseph HypoliteJoseph Hypolite Glenolden, PA More articles by this author , Qi LeiQi Lei Glenolden, PA More articles by this author , Shaohua ChangShaohua Chang Glenolden, PA More articles by this author , Anna MalykhinaAnna Malykhina Glenolden, PA More articles by this author , Stephen ZdericStephen Zderic Philadelphia, PA More articles by this author , Alan WeinAlan Wein Philadelphia, PA More articles by this author , Stephan ButlerStephan Butler Philadelphia, PA More articles by this author , and Samuel ChackoSamuel Chacko Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2154AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Partial bladder outlet obstruction (PBOO)-induced detrusor smooth muscle hypertrophy is associated with a downregulation of protein kinase C (PKC) and large conductance calcium-activated potassium channels (BK) channels, and an increase in spontaneous contractions in isolated muscle strips, and non-voiding contractions in patients with BPH-induced PBOO. The purpose of this study is to examine whether or not PKC is involved in the regulation of spontaneous and non-voiding contractions in DSM, and whether inhibition of PKC contributes to increased detrusor excitability. METHODS DSM strips (∼2mm × 10mm) were removed from 2-week PBOO rabbits and used for in vitro contractile studies. Sham-operated animals served as controls. The effect of inhibition of PKC on non-voiding contractions was studied using in vivo cystometry in rats, and whole cell patch clamping was used to study the effect of phorbol 12,13-dibutyrate (PDBU), a PKC activator, on the BK channel current. Iberiotoxin (IBX), a BK-channel inhibitor, was used to study its effect on spontaneous contractions and PDBU-induced inhibition of spontaneous contraction. RESULTS The results indicate that the PKC inhibitor, bisindolylmaleimide1, (BIS-1) and IBX increased the amplitude of spontaneous contractions in detrusor smooth muscle strips from normal rabbit bladders but that there was no significant change in the strips from PBOO. BIS-1 also increased the frequency of non-voiding contractions in normal rat bladder. Both BIS-1- and IBX-induced spontaneous contractions were inhibited by PDBU (10-50 nM). BIS-1-induced spontaneous contractions were significantly reduced by BK channel openers, NS1619 and isopimeric acid. Pre-treatment of isolated muscle strips with IBX significantly delayed the inhibitory effect of PDBU on spontaneous contractions. Whole cell patch clamp studies revealed that PDBU caused an increase in the BK channel current in detrusor myocytes isolated from both sham-operated and PBOO animals. CONCLUSIONS The data suggest that PKC may play an important role in maintaining the relatively quiescent, non-excitable state of the normal bladder by suppressing spontaneous and non-voiding contractions, and that down-regulation of PKC in PBOO may contribute to detrusor overactivity. The data also suggests that the mechanism of action may involve the ability of PKC to increase the BK-channel current during the filling phase of the micturition cycle. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e780 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joseph Hypolite Glenolden, PA More articles by this author Qi Lei Glenolden, PA More articles by this author Shaohua Chang Glenolden, PA More articles by this author Anna Malykhina Glenolden, PA More articles by this author Stephen Zderic Philadelphia, PA More articles by this author Alan Wein Philadelphia, PA More articles by this author Stephan Butler Philadelphia, PA More articles by this author Samuel Chacko Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Regulation of Prostate Cancer Cell Survival by Protein Kinase C∈ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.

Disruption of noncanonical Wnt/CA 2+ pathway in the cadmium-induced omphalocele in the chick model

Purpose Cadmium (Cd) has been found to cause ventral body wall defects (VBWDs) in the chick embryo similar to human omphalocele. The earliest detectable histologic changes in Cd-induced VBWD chick model have been observed 4 hours posttreatment. The exact mechanism by which Cd acts in the early embryogenesis remains unclear. Wnt proteins play a key role during embryogenesis, and altered Wnt signaling has been linked to developmental defects. Noncanonical Wnt/Ca 2+ pathway has been implicated in regulating embryogenesis by controlling cell movement and adhesion. Wnt11 can activate protein kinase C (PKC) and calcium/calmodulin-dependent kinase II (CaMKII) in the Wnt/Ca 2+ pathway. We hypothesized that the Wnt11, PKC α, and CaMKII gene expression is downregulated in the Cd-induced VBWD during early embryogenesis. Methods After 60 hours of incubation, chick embryos were harvested 1 hour (1H), 4H, and 8H after treatment of saline or cadmium and divided into 2 groups: control and Cd (n = 8 at each time-point, respectively). Real-time polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression of Wnt11, PKC α, and CaMKII in the Cd-induced VBWD chick model. Results The mRNA expression levels of Wnt11, PKC α, and CaMKII were significantly decreased at 1H in Cd group compared to controls ( P < .05). However, there were no significant differences in the other time-points. Conclusion Downregulation of Wnt11, PKC α, and CaMKII gene expression during the narrow window of early embryogenesis may cause VBWD, interfering with cell movement and adhesion, disrupting Wnt/Ca 2+ pathway.