PKCα Activation Research Articles

Curcumin exerts hepatoprotection via overexpression of Paraoxonase-1 and its regulatory genes in rats undergone bile duct ligation.

Curcumin is described as an antioxidant, hepato-protective and antifibrotic in liver fibrosis, although its mechanism is still not known. One of the models of the chronic liver disease stemming from oxidative stress and the generation of free radical has been considered to be bile duct ligation (BDL). Paraoxonase 1 (PON1) is a prominent antioxidant enzyme. Therefore, the objective of the present research is to assess the effects of curcumin on upregulation of PON1 in BDL rats. As predicted, the rats have been divided into the four groups of Sham, Sham+Cur (curcumin), BDL and BDL+Cur. We evaluated the efficacy of curcumin (100mg/kg/day) on protein and gene expression of PON1 and regulatory genes contributed to the gene expression PON1 such as Sp1, PKCα, SREBP-2, AhR, JNK and regulation PON1 activity gene expression of Apo A1. Curcumin attenuated alterations in liver histology, hepatic enzymes and the mRNA expression of fibrotic markers (p<0.05). In addition, curcumin increased significantly mRNA, protein expression of PON1 and mRNA of the genes that are contributed to the expression of PON1 such as Sp1, PKCα, SREBP-2, AhR, JNK and increased PON1 activity through upregulation of Apo A1 (p<0.05). Cirrhosis progression may be inhibited by treatment with curcumin through the increased influence the expression and activity of PON1.

Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2.

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there isno information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.

Protein Kinase C Alpha Cellular Distribution, Activity, and Proximity with Lamin A/C in Striated Muscle Laminopathies.

Striated muscle laminopathies are cardiac and skeletal muscle conditions caused by mutations in the lamin A/C gene (LMNA). LMNA codes for the A-type lamins, which are nuclear intermediate filaments that maintain the nuclear structure and nuclear processes such as gene expression. Protein kinase C alpha (PKC-α) interacts with lamin A/C and with several lamin A/C partners involved in striated muscle laminopathies. To determine PKC-α’s involvement in muscular laminopathies, PKC-α’s localization, activation, and interactions with the A-type lamins were examined in various cell types expressing pathogenic lamin A/C mutations. The results showed aberrant nuclear PKC-α cellular distribution in mutant cells compared to WT. PKC-α activation (phos-PKC-α) was decreased or unchanged in the studied cells expressing LMNA mutations, and the activation of its downstream targets, ERK 1/2, paralleled PKC-α activation alteration. Furthermore, the phos-PKC-α-lamin A/C proximity was altered. Overall, the data showed that PKC-α localization, activation, and proximity with lamin A/C were affected by certain pathogenic LMNA mutations, suggesting PKC-α involvement in striated muscle laminopathies.

A metabolic reaction-diffusion model for PKCα translocation via PIP2 hydrolysis in an endothelial cell.

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) at the cell membrane induces the release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm and diffusion of diacylglycerol (DAG) through the membrane, respectively. Release of IP3 subsequently increases Ca2+ levels in the cytoplasm, which results in activation of protein kinase C α (PKCα) by Ca2+ and DAG, and finally the translocation of PKCα from the cytoplasm to the membrane. In this study, we developed a metabolic reaction-diffusion framework to simulate PKCα translocation via PIP2 hydrolysis in an endothelial cell. A three-dimensional cell model, divided into membrane and cytoplasm domains, was reconstructed from confocal microscopy images. The associated metabolic reactions were divided into their corresponding domain; PIP2 hydrolysis at the membrane domain resulted in DAG diffusion at the membrane domain and IP3 release into the cytoplasm domain. In the cytoplasm domain, Ca2+ was released from the endoplasmic reticulum, and IP3, Ca2+, and PKCα diffused through the cytoplasm. PKCα bound Ca2+ at, and diffused through, the cytoplasm, and was finally activated by binding with DAG at the membrane. Using our model, we analyzed IP3 and DAG dynamics, Ca2+ waves, and PKCα translocation in response to a microscopic stimulus. We found a qualitative agreement between our simulation results and our experimental results obtained by live-cell imaging. Interestingly, our results suggest that PKCα translocation is dominated by DAG dynamics. This three-dimensional reaction-diffusion mathematical framework could be used to investigate the link between PKCα activation in a cell and cell function.

Adenosine Triphosphate Accumulated Following Cerebral Ischemia Induces Neutrophil Extracellular Trap Formation.

In ischemic stroke, neutrophils infiltrate damaged brain tissue immediately following the ischemic insult and aggravate inflammation via various mechanisms which include neutrophil extracellular traps (NETs) formation. In the present study, we showed that adenosine triphosphate (ATP), a DAMP molecule, accumulates in the brain and induces NETosis in brain parenchyma and in circulating neutrophils (PMNs) isolated from a murine model of stroke induced by middle cerebral artery occlusion (MCAO). Expression of peptidylarginine deiminase-4 (PAD4), which induces citrullination of histones H3 (CitH3) and initiates NETosis, was significantly enhanced in brain parenchyma and blood PMNs following MCAO. ATP or BzATP (a prototypic P2X7R agonist) significantly enhanced the inductions of PAD4 and CitH3 in a P2X7R-dependent manner and intracellular Ca2+ influx, PKCα activation, and NADPH oxidase-dependent reactive oxygen species (ROS) production play critical roles in this ATP-P2X7R-mediated NETosis. In our MCAO animal model, NETosis was markedly suppressed by treatment with apyrase, an enzyme hydrolyzing ATP, but enhanced by co-treatment of BzATP, confirming ATP-P2X7R-mediated NETosis. Since ATP not only induced NETosis but was also extruded after NETosis, our results indicate that ATP accumulated in the ischemic brain induces NETosis, mediating a cross-talk linking NETosis with neuronal damage that might aggravate inflammation and brain damage.

Activation of PKCα participates in the reduction of Ikur in atrial myocytes induced by tumour necrosis factor-α.

The atrial-specific ultra-rapid delayed rectifier K+ current (Ikur) plays an important role in the progression of atrial fibrillation (AF). Because inflammation is known to lead to the onset of AF, we aimed to investigate whether tumour necrosis factor-α (TNF-α) played a role in regulating Ikur and the potential signalling pathways involved. Whole-cell patch-clamp and biochemical assays were used to study the regulation and expression of Ikur in myocytes and in tissues from left atrial appendages (LAAs) obtained from patients with sinus rhythm (SR) or AF, as well as in rat cardiomyocytes (H9c2 cells) and mouse atrial myocytes (HL-1 cells). Ikur current density was markedly reduced in atrial myocytes from AF patients compared with SR controls. Reduction of Kv1.5 protein levels was accompanied by increased expression of TNF-α and protein kinase C (PKC)α activation in AF patients. Treatment with TNF-α dose-dependently reduced Ikur and protein expression of Kv1.5 but not Kv3.1b in H9c2 cells and HL-1 cells. TNF-α also increased activity of PKCα. Specific PKCα inhibitor Gö6976 alleviated the reduction in Ikur induced by TNF-α, but not the reduction in Kv1.5 protein. TNF-α was involved in the electrical remodelling associated with AF, probably by depressing Ikur in atrial myocytes via activation of PKCα.

Dexmedetomidine Attenuates LPS-Induced Monocyte-Endothelial Adherence via Inhibiting Cx43/PKC-α/NOX2/ROS Signaling Pathway in Monocytes.

Dexmedetomidine is widely used for sedating patients in operation rooms or intensive care units. Its protective functions against oxidative stress, inflammation reaction, and apoptosis have been widely reported. In present study, we explored the effects of dexmedetomidine on monocyte-endothelial adherence. We built lipopolysaccharide- (LPS-) induced monocyte-endothelial adherence models with U937 monocytes and human umbilical vein endothelial cells (HUVECs) and observed the effects of dexmedetomidine on U937-HUVEC adhesion. Specific siRNA was designed to knock-down Connexin43 (Cx43) expression in U937 monocytes. Gö6976, GSK2795039, and NAC were used to inhibit PKC-α, NOX2, and ROS, respectively. Then, we detected whether dexmedetomidine could downregulate Cx43 expression and its downstream PKC-α/NOX2/ROS signaling pathway activation and ultimately result in the decrease of U937-HUVEC adhesion. The results showed that dexmedetomidine, at its clinically relevant concentrations (0.1 nM and 1 nM), could inhibit adhesion of molecule expression (VLA-4 and LFA-1) and U937-HUVEC adhesion. Simultaneously, it also attenuated Cx43 expression in U937 monocytes. With the downregulation of Cx43 expression, the activity of PKC-α and its related NOX2/ROS signaling pathway were reduced. Inhibiting PKC-α/NOX2/ROS signaling pathway with Gö6976, GSK2795039, and NAC, respectively, VLA-4, LFA-1 expression, and U937-HUVEC adhesion were all decreased. In summary, we concluded that dexmedetomidine, at its clinically relevant concentrations (0.1 nM and 1 nM), decreased Cx43 expression in U937 monocytes and PKC-α associated with carboxyl-terminal domain of Cx43 protein. With the downregulation of PKC-α, the NOX2/ROS signaling pathway was inhibited, resulting in the decrease of VLA-4 and LFA-1 expression. Ultimately, U937-HUVEC adhesion was reduced.

The role of protein kinase C alpha in tri-ortho-cresyl phosphate-induced autophagy in human neuroblastoma SK-N-SH cells.

As an organophosphorus ester, tri-ortho-cresyl phosphate (TOCP) has been widely used in agriculture and industry. It is reported that TOCP can induce organophosphate-induced delayed neuropathy (OPIDN) in sensitive animal and human species. However, the exact molecular mechanisms underlying TOCP-induced neurotoxicity are still unknown. In this study, we found that TOCP could induce autophagy by activating protein kinase C alpha (PKCα) signaling in neuroblastoma SK-N-SH cells. PKCα activators could positively regulate TOCP-induced autophagy by increasing the expression levels of neighbor BRCA1 gene protein 1 (NBR1), LC3 and P62 autophagic receptor protein. Furthermore, PKCα activation impaired the ubiquitin-proteasome system (UPS), resulting in inhibition of proteasome activity and accumulation of ubiquitinated proteins. UPS dysfunction could stimulate autophagy to serve as a compensatory pathway, which contributed to the accumulation of the abnormally hyperphosphorylated tau proteins and degradation of impaired proteins of the MAP 2 and NF-H families in neurodegenerative disorders.

Nrf2/HO-1 partially regulates cytoprotective effects of carbon monoxide against urban particulate matter-induced inflammatory responses in oral keratinocytes

IntroductionExposure to airborne particulate matter (PM) increases the proportion of oral inflammatory diseases. During the formation of inflammatory conditions, the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome activation plays an important regulator. Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by heme oxygenase-1 (HO-1), has been shown to own cytoprotective effects including anti-inflammation and antioxidant. Here, we determined the novel mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on PM-induced inflammatory responses in human oral keratinocytes (HOKs). MethodsThe effects of CORM-2 on the expression of various inflammatory proteins induced by PM were determined by Western blot, real-time PCR, promoter assay, and ELISA. The involvement of signaling molecules in these responses was studied by using the selective pharmacological inhibitors and siRNAs. ResultsWe proved that PM enhanced C-reactive protein (CRP) levels, NLRP3 inflammasome and caspase-1 activation, and IL-1β release, which were reduced by preincubation with CORM-2. Transfection with PKCα siRNA and preincubation with the ROS scavenger (N-acetyl-cysteine, NAC), an inhibitor of NADPH oxidase (diphenyleneiodonium, DPI), or the mitochondria-specific superoxide scavenger (MitoTEMPO) inhibited PM-mediated inflammatory responses. In addition, PM-regulated PKCα and NADPH oxidase activation as well as NADPH oxidase- and mitochondria-derived ROS generation were inhibited by CORM-2, but not inactivate CORM-2 (iCORM-2) pretreatment. At the end, we confirmed that CORM-2 improved PM-induced inflammatory responses via the induction of Nrf2 activation and HO-1 expression. ConclusionWe suggest that CORM-2 inhibits PM-induced inflammatory responses in HOKs via the inhibition of PKCα/ROS/NLRP3 inflammasome activation combined with the induction of Nrf2/HO-1 expression.

Preclinical and Clinical Studies on Bryostatins, A Class of Marine-Derived Protein Kinase C Modulators: A Mini-Review

Bryostatins are complex macrolactones isolated from marine organisms Bryozoan Bugula neritina. They are potent modulators of protein kinase C isozymes (PKCα: ki = 1.3-188 nM), and are one of the most extensively investigated marine natural products in clinical trials. Although ~21 natural bryostatins have been isolated, however only bryostatin-1 (1) has received much interest among medicinal chemists and clinicians. The structure-activity relationship of bryostatins has been well established, with the identification of key pharmacophoric features important for PKC modulation. The low natural abundance and the long synthetic route have prompted medicinal chemists to come-up with simplified analogs. Bryostatin skeleton comprises three pyran rings connected to each other to form a macrocyclic lactone. The simplest analog 27 contains only one pyran, which is also able to modulate the PKCα activity; however, the cyclic framework appears to be essential for the desired level of potency. Another simplified analog 17 ("picolog") exhibited potent and in-vivo efficacy against lymphoma. Bryostatin-1 (1) has shown an acceptable intravenous pharmacokinetic profile in mice and displayed promising in-vivo efficacy in mice models of various cancers and Alzheimer's disease. Bryostatin-1 was investigated in numerous Phase I/II oncology clinical trials; it has shown minimal effect as a single agent, however, provided encouraging results in combination with other chemotherapy agents. FDA has granted orphan drug status to bryostatin-1 in combination with paclitaxel for esophageal cancer. Bryostatin-1 has also received orphan drug status for fragile X syndrome. Bryostatin-1 was also investigated in clinical studies for Alzheimer's disease and HIV infection. In a nutshell, the natural as well as synthetic bryostatins have generated a strong hope to emerge as treatment for cancer along with many other diseases.

Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules.

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II's ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-β, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (-15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (-0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-β in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min-1·mg protein-1 in fructose group, and the PKC-α/β inhibitor Gö6976 blocked this increase (-96 ± 205 nmol·min-1·mg protein-1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Titin and CK2α are New Intracellular Targets in Acute Insulin Application-Associated Benefits on Electrophysiological Parameters of Left Ventricular Cardiomyocytes From Insulin-Resistant Metabolic Syndrome Rats.

Previous studies have demonstrated that a high-carbohydrate intake could induce metabolic syndrome (MetS) in male rats with marked cardiac functional abnormalities. In addition, studies mentioned some benefits of insulin application on these complications, but there are considerable disagreements among their findings. Therefore, we aimed to extend our knowledge on the in-vitro influence of insulin on left ventricular dysfunction and also in the isolated cardiomyocytes from MetS rats. At the organ function level, an acute insulin application (100-nM) provided an important beneficial effect on the left ventricular developed pressure in MetS rats. Furthermore, to treat the freshly isolated cardiomyocytes from MetS rats with insulin provided marked recoveries in elevated resting intracellular Ca2+-level, as well as significant prevention of prolonged action potential through an augmentation in depressed K+-channel currents. Insulin also normalized the cellular levels of increased ROS and phosphorylation of PKCα, together with normalizations of apoptotic markers in MetS cardiomyocytes through the insulin-mediated regulation of phospho-Akt. Since not only elevated PKCα-activity but also reductions in phospho-Akt are key modulators of titin-based cardiomyocyte stiffening in hyperglycemia, insulin treatment of the cardiomyocytes prevented the activation of titin via the above pathways. Furthermore, CK2α-activation and NOS-phosphorylation could be prevented with insulin treatment. Mechanistically, we found that impaired insulin signaling and elevated PKCα and CK2α activities, as well as depressed Akt phosphorylation, are key modulators of titin-based cardiomyocyte stiffening in MetS rats. We propose that restoring normal kinase activities and also increases in phospho-Akt by insulin can contribute marked recoveries in MetS heart function, indicating a promising approach to modulate titin-associated factors in heart dysfunction associated with type-2 diabetes mellitus. Graphical Abstract.

Increase in PKCα Activity during Heart Failure Despite the Stimulation of PKCα Braking Mechanism.

Rationale: Heart failure (HF) is marked by dampened cardiac contractility. A mild therapeutic target that improves contractile function without desensitizing the β-adrenergic system during HF may improve cardiac contractility and potentially survival. Inhibiting protein kinase C α (PKCα) activity may fit the criteria of a therapeutic target with milder systemic effects that still boosts contractility in HF patients. PKCα activity has been observed to increase during HF. This increase in PKCα activity is perplexing because it is also accompanied by up-regulation of a molecular braking mechanism. Objective: I aim to explore how PKCα activity can be increased and maintained during HF despite the presence of a molecular braking mechanism. Methods and Results: Using a computational approach, I show that the local diacylglycerol (DAG) signaling is regulated through a two-compartment signaling system in cardiomyocytes. These results imply that after massive myocardial infarction (MI), local homeostasis of DAG signaling is disrupted. The loss of this balance leads to prolonged activation of PKCα, a key molecular target linked to LV remodeling and dysfunctional filling and ejection in the mammalian heart. This study also proposes an explanation for how DAG homeostasis is regulated during normal systolic and diastolic cardiac function. Conclusions: I developed a novel two-compartment computational model for regulating DAG homeostasis during Ang II-induced heart failure. This model provides a promising tool with which to study mechanisms of DAG signaling regulation during heart failure. The model can also aid in identification of novel therapeutic targets with the aim of improving the quality of life for heart failure patients.

Cytosolic Ca2+ modulates Golgi structure through PKC‐mediated GRASP55 phosphorylation

It has been well documented that the endoplasmic reticulum (ER) responds to cellular stresses through the unfolded protein response (UPR), but it is unknown how the Golgi apparatus responds to similar stresses. In this study, we treated HeLa cells with ER stress inducers, thapsigargin (TG), tunicamycin (Tu) and Dithiothreitol (DTT), and found that only TG treatment resulted in Golgi fragmentation. TG induced Golgi fragmentation at a low dose and short time when UPR was undetectable, demonstrating that Golgi fragmentation occurs independently of ER stress. Further experiments demonstrated that Golgi fragmentation was through elevated intracellular Ca2+ and protein kinase Cα (PKCα) activity, which phosphorylates the Golgi stacking protein GRASP55. Significantly, activation of PKCα with other activating or inflammatory agents, including Phorbol 12‐myristate 13‐acetate (PMA) and histamine, modulates the Golgi structure in a similar fashion. Hence, our study revealed a novel mechanism through which increased cytosolic Ca2+ modulates Golgi structure and function.Support or Funding InformationThis work was supported by the National Institutes of Health (Grants GM112786, GM105920, and GM130331), MCubed and the Fastforward Protein Folding Disease Initiative of the University of Michigan to Y. Wang. S. Ireland is a graduate student who has been partially supported by the Mary Sue and Kenneth Coleman Endowed Fellowship Fund from the University of Michigan.

Tetraspanin CD82 drives acute myeloid leukemia chemoresistance by modulating protein kinase C alpha and β1 integrin activation.

A principal challenge in treating acute myeloid leukemia (AML) is chemotherapy refractory disease. As such, there remains a critical need to identify key regulators of chemotherapy resistance in AML. In this study, we demonstrate that the membrane scaffold, CD82, contributes to the chemoresistant phenotype of AML. Using an RNA-seq approach, we identified the increased expression of the tetraspanin family member, CD82, in response to the chemotherapeutic, daunorubicin. Analysis of the TARGET and BEAT AML databases identifies a correlation between CD82 expression and overall survival of AML patients. Moreover, using a combination of cell lines and patient samples, we find that CD82 overexpression results in significantly reduced cell death in response to chemotherapy. Investigation of the mechanism by which CD82 promotes AML survival in response to chemotherapy identified a crucial role for enhanced protein kinase c alpha (PKCα) signaling and downstream activation of the β1 integrin. Additionally, analysis of β1 integrin clustering by super-resolution imaging demonstrates that CD82 expression promotes the formation of dense β1 integrin membrane clusters. Lastly, evaluation of survival signaling following daunorubicin treatment identified robust activation of p38 mitogen-activated protein kinase (MAPK) downstream of PKCα and β1 integrin signaling when CD82 is overexpressed. Together, these data propose a mechanism where CD82 promotes chemoresistance by increasing PKCα activation and downstream activation/clustering of β1 integrin, leading to AML cell survival via activation of p38 MAPK. These observations suggest that the CD82-PKCα signaling axis may be a potential therapeutic target for attenuating chemoresistance signaling in AML.

Cytosolic Ca2+ Modulates Golgi Structure Through PKCα-Mediated GRASP55 Phosphorylation.

SummaryIt has been well documented that the ER responds to cellular stresses through the unfolded protein response (UPR), but it is unknown how the Golgi responds to similar stresses. In this study, we treated HeLa cells with ER stress inducers, thapsigargin (TG), tunicamycin (Tm), and dithiothreitol (DTT), and found that only TG treatment resulted in Golgi fragmentation. TG induced Golgi fragmentation at a low dose and short time when UPR was undetectable, indicating that Golgi fragmentation occurs independently of ER stress. Further experiments demonstrated that TG induces Golgi fragmentation through elevating intracellular Ca2+ and protein kinase Cα (PKCα) activity, which phosphorylates the Golgi stacking protein GRASP55. Significantly, activation of PKCα with other activating or inflammatory agents, including phorbol 12-myristate 13-acetate and histamine, modulates Golgi structure in a similar fashion. Hence, our study revealed a novel mechanism through which increased cytosolic Ca2+ modulates Golgi structure and function.

Evaluation of Toxic Amyloid 42 Oligomers in Rat Primary Cerebral Cortex Cells and Human iPS-derived Neurons Treated with 10-Me-Aplog-1, a New PKC Activator.

Amyloid β42 (Aβ42), a causative agent of Alzheimer’s disease (AD), is derived extracellularly from Aβ precursor protein (APP) following the latter’s cleavage by β-secretase, but not α-secretase. Protein kinase Cα (PKCα) activation is known to increase α-secretase activity, thereby suppressing Aβ production. Since Aβ42 oligomer formation causes potent neurotoxicity, APP modulation by PKC ligands is a promising strategy for AD treatment. Although bryostatin-1 (bryo-1) is a leading compound for this strategy, its limited natural availability and the difficulty of its total synthesis impedes further research. To address this limitation, Irie and colleagues have developed a new PKC activator with few side effects, 10-Me-Aplog-1, (1), which decreased Aβ42 in the conditioned medium of rat primary cerebral cortex cells. These results are associated with increased α-secretase but not PKCε-dependent Aβ-degrading enzyme. The amount of neuronal embryonic lethal abnormal vision (nELAV), a known β-secretase stabilizer, was reduced by treatment with 1. Notably, 1 prevented the formation of intracellular toxic oligomers. Furthermore, 1 suppressed toxic oligomerization within human iPS-derived neurons such as bryo-1. Given that 1 was not neurotoxic toward either cell line, these findings suggest that 1 is a potential drug lead for AD therapy.

Role of PKCζ-NADPH oxidase signaling axis in PKCα-mediated Giα2 phosphorylation for inhibition of adenylate cyclase activity by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C-ζ (PKC-ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC-α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC-ζ, NADPH oxidase, and PKC-α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC-ζ and NADPH oxidase attenuate AngII induced increase in PKC-α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC-α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC-ζ and NADPH oxidase activities. Overall, PKCζ-NADPH oxidase-PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII-mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs.

Five Constituents in Psoralea corylifolia L. Attenuate Palmitic Acid-Induced Hepatocyte Injury via Inhibiting the Protein Kinase C-α/Nicotinamide-Adenine Dinucleotide Phosphate Oxidase Pathway.

Psoralea corylifolia L. (PC) is a traditional Chinese herb used to treat yang deficiency of the spleen and kidney in pediatric disease. Our previous studies have found that PC can alleviate the liver oxidative stress of juvenile mice with nonalcoholic steatohepatitis (NASH), and its mechanism is related to the inhibition of the protein kinase C-α (PKC-α)/nicotinamide-adenine dinucleotide phosphate oxidase (NOX) signaling pathway. The aim of this study was to confirm the aforementioned drug target in vitro and to conduct preliminary screening for some effective compounds of PC on the treatment of NASH. A primary hepatocyte model of non alcoholic fatty liver disease was established by palmitic acid. The existence of Psoralen, Isopsoralen, Neobavaisoflavone, Isobavachalcone, and Bakuchiol were identified by ultra-performance liquid chromatography. Then, five PC compounds were administered. Intracellular triglyceride and total cholesterol content, the cell supernatant alanine aminotransferase and aspartate aminotransferase, and hepatocellular superoxide anion were examined. The changes of PKC-α/NOX signaling pathways in hepatocytes were also determined. Furthermore, PKC-α activator phorbol 12-myristate 13-acetate was administered for 4 h before Psoralen intervention was conducted again to detect the changes of PKC-α/NOX signaling pathways. Our data demonstrated that Psoralen, Isopsoralen, and Isobavachalcone decreased intracellular content of triglyceride while all five PC compounds improved hepatocellular total cholesterol accumulation and hepatocyte damage in palmitic acid-induced primary hepatocyte model of non alcoholic fatty liver disease. All five PC compounds could also reduce hepatocytic superoxide anion levels, nicotinamide-adenine dinucleotide phosphate/reduced nicotinamide-adenine dinucleotide phosphate ratio, NOX activity as well as p47phox protein expression and PKCα activation in hepatocytes. Psoralen exhibited the best efficacy but the effectiveness was lost when pre-stimulated by phorbol 12-myristate 13-acetate. The results suggest that Psoralen, Isopsoralen, and Isobavachalcone could improve hepatocyte steatosis; five PC compounds could ameliorate hepatocyte injury, relieve oxidative stress, and downregulate the PKC-α/NOX signaling pathway of hepatocytes. In addition, Psoralen exhibits the best efficacy and a prospective PKC-α inhibitor pharmaceutical activity.

Metoprolol and bisoprolol ameliorate hypertrophy of neonatal rat cardiomyocytes induced by high glucose via the PKC/NF-κB/c-fos signaling pathway.

Hyperglycemia caused by diabetes mellitus could increase the risk of diabetic cardiomyopathy. However, to the best of our knowledge, the underlying mechanism of this process is still not fully explored. Thus, developing ways to prevent hyperglycemia can be beneficial for diabetic patients. The present study was designed to investigate the influence of metoprolol and bisoprolol on the cardiomyocytic hypertrophy of neonatal rat cardiomyocytes. Cardiomyocytes were cultured in two types of media: One with low glucose levels and one with high glucose levels. Cardiomyocytes cultured in high glucose were further treated with the following: A protein kinase C (PKC) inhibitor, an NF-κB inhibitor, metoprolol or bisoprolol. The pulsatile frequency, cellular diameter and surface area of cardiomyocytes were measured. Protein content and [3H]-leucine incorporation were determined, atrial natriuretic peptide (ANP), α-myosin heavy chain (α-MHC) and β-myosin heavy chain (β-MHC) mRNA levels were calculated by reverse transcription-quantitative PCR, while the expression and activation of PKC-α, PKC-β2, NF-κB, tumor necrosis factor-α (TNF-α), and c-fos were detected by western blotting. Metoprolol or bisoprolol were also used in combination with PKC inhibitor or NF-κB inhibitor to determine whether the hypertrophic response would be attenuated to a lower extent compared with metroprolol or bisoprolol alone. Cardiomyocytes cultured in high glucose presented increased pulsatile frequency, cellular diameter, surface area, and protein content and synthesis, higher expression of ANP and β-MHC, and lower α-MHC expression. High glucose levels also upregulated the expression and activation of PKC-α, PKC-β2, NF-κB, TNF-α and c-fos. Metoprolol and bisoprolol partly reversed the above changes, while combined use of metoprolol or bisoprolol with PKC inhibitor or NF-κB inhibitor further ameliorated the hypertrophic response mentioned above to lower levels compared with using metroprolol or bisoprolol alone. In conclusion, metoprolol and bisoprolol could prevent hypertrophy of cardiomyocytes cultured in high glucose by the inhibition of the total and phospho-PKC-α, which could further influence the PKC-α/NF-κB/c-fos signaling pathway.