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Protein kinase C epsilon (PKCε) plays important roles in behavioral responses to alcohol and in anxiety-like behavior in rodents, making it a potential drug target for reducing alcohol consumption and anxiety. Identifying signals downstream of PKCε could reveal additional targets and strategies for interfering with PKCε signaling. We used a chemical genetic screen combined with mass spectrometry to identify direct substrates of PKCε in mouse brain, and validated findings for 39 of them using peptide arrays and in vitro kinase assays. Prioritizing substrates with several public databases such as LINCS-1000, STRING, GeneFriends, and GeneMAINA, predicted interactions between these putative substrates and PKCε, and identified substrates associated with alcohol-related behaviors, actions of benzodiazepines, and chronic stress. The 39 substrates could be broadly classified in three functional categories: cytoskeletal regulation, morphogenesis, and synaptic function. These results provide a list of brain PKCε substrates, many of which are novel, for future investigation to determine the role of PKCε signaling in alcohol responses, anxiety, responses to stress, and other related behaviors.

Ischemic strokes are associated with a high rate of disability and death globally. Cerebral ischemia/reperfusion (I/R) injury is a type of brain damage associated with oxidative stress after an ischemic stroke. Beta-boswellic acid (β-BA) reportedly exerts antioxidant and neuroprotective effects, but its role in cerebral I/R injury is unclear. The aim of this research was to investigate the neuroprotective effects, as well as the mechanisms of β-BA in cerebral I/R injury. In vivo experiments were conducted using a rat middle cerebral artery occlusion and reperfusion (MCAO/R) model, and in vitro experiments were performed using a rat neuronal oxygen-glucose deprivation and reoxygenation (OGD/R) model. Triphenyltetrazolium chloride staining, neurological function scores, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, hematoxylin and eosin staining, and antioxidant levels in the brain were used to assess the effects of β-BA. Flow cytometry was used to detect reactive oxygen species and apoptotic cells. Western blotting and immunofluorescence staining were used to measure protein levels. The results showed that β-BA markedly improved neurological deficits and decreased infarct volume and necrotic neurons in rats. The in vitro results showed that β-BA protected neurons against OGD/R-induced injury. Additionally, β-BA significantly increased the phosphorylation of protein kinase C epsilon (PRKCE) at S729, the translocation of nuclear factor erythroid 2-like 2 (NFE2L2), and expression of heme oxygenase-1 (HMOX1). This study demonstrates that β-BA exerts neuroprotective effects against cerebral I/R via the activation of the PRKCE/NFE2L2/HMOX1 pathway and is a potential therapeutic candidate for ischemic stroke.

Background Optimal non-invasive biomarkers for diagnosis and treatment of nonalcoholic fatty liver disease (NAFLD) remain to be identified. Aims To identify potential DNA methylation biomarkers for NAFLD. Methods Genome-wide DNA methylation profiling was performed to identify differentially methylated CpG sites in peripheral blood leukocytes. Differentially methylated regions were validated using the MassCLEAVE assay. The expression levels of candidate genes were explored by Gene Expression Omnibus database. Results The hypomethylation of PRKCE CpG 4.5 and CpG 18.19 was associated with nonalcoholic fatty liver (NAFL), the odds ratio (OR) and 95% confidence interval (CI) were 0.129 (0.026–0.639) and 0.231 (0.069–0.768). The methylation level of CpG 1.2 and average methylation level of SEC14L3 were correlated with NAFL, with OR (95% CI) being 0.283 (0.093–0.865) and 0.264 (0.087–0.799). PRKCE CpG 4.5 and cg17802464 of SEC14L3 were correlated with body mass index, waist circumference, total triglyceride, high-density lipoprotein cholesterol, alanine aminotransferase and aspartate aminotransferase. All selected datasets showed high expression levels of PRKCE and SEC14L3 in patients with NAFLD. Conclusions Our findings suggest that the hypomethylation of PRKCE and SEC14L3 promoters represent attractive biomarkers for NAFLD. Further studies are warranted to validate these biomarkers as molecular tools for diagnosis of NAFLD and therapeutic targets.

Novel protein kinase C (nPKC) family member, protein kinase C epsilon (PKCε) is an AGC kinase superfamily member. It is associated with neurological and metabolic diseases as well as human cancers. No study so far has been conducted to identify genetic variations and their effect on PKCε folding and functioning. The present study aimed to identify mutational hotspots in PKCε and disease-causing non-synonymous variants (nsSNPs) along with the investigation of nsSNP impact on protein dynamics. Twenty-nine in silico tools were applied to determine nsSNP deleteriousness, their impact on protein dynamics and disease association, along with the prediction of PKCε post-translational modification (PTM) sites. The present study’s outcomes indicated that most nsSNPs were concentrated in the PKCε hinge region and C-terminal tail. Most pathogenic variants mapped to the kinase domain. Regulatory domain variants influenced PKCε interaction with molecular players whereas kinase domain variants were predicted to impact its phosphorylation pattern and protein–protein interactions. Most PTM sites were mapped to the hinge region. PKCε nsSNPs have an association with oncogenicity and its expression dysregulation is responsible for poor overall survival. Understanding nsSNP structural impact is a primary step necessary for delineating the relationship of genetic level differences with protein phenotype. The obtained knowledge can eventually help in disease diagnosis and therapy design.

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P 2 ). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP 2 , an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

Transient receptor potential vanillic acid 1 (TRPV1) is an ion channel activated by heat and inflammatory factors involved in the development of various types of pain. The P2X7 receptor is in the P2X family and is associated with pain mediated by satellite glial cells. There might be some connection between the P2X7 receptor and TRPV1 in neuropathic pain in diabetic rats. A type 2 diabetic neuropathic pain rat model was induced using high glucose and high-fat diet for 4 weeks and low-dose streptozocin (35 mg/kg) intraperitoneal injection to destroy islet B cells. Male Sprague Dawley rats were administrated by intrathecal injection of P2X7 shRNA and p38 inhibitor, and we recorded abnormal mechanical and thermal pain and nociceptive hyperalgesia. One week later, the dorsal root ganglia from the L4-L6 segment of the spinal cord were harvested for subsequent experiments. We measured pro-inflammatory cytokines, examined the relationship between TRPV1 on neurons and P2X7 receptor on satellite glial cells by measuring protein and transcription levels of P2X7 receptor and TRPV1, and measured protein expression in the PKCε/P38 MAPK/NF-κB signaling pathway after intrathecal injection. P2X7 shRNA and p38 inhibitor relieved hyperalgesia in diabetic neuropathic pain rats and modulated inflammatory factors in vivo. P2X7 shRNA and P38 inhibitors significantly reduced TRPV1 expression by downregulating the PKCε/P38 MAPK/NF-κB signaling pathway and inflammatory factors in dorsal root ganglia. Intrathecal injection of P2X7 shRNA alleviates nociceptive reactions in rats with diabetic neuropathic pain involving TRPV1 via PKCε/P38 MAPK/NF-κB signaling pathway.

N, N-Dimethylformamide (DMF) can cause liver damage in occupationally exposed workers, but the molecular mechanism of DMF-induced liver damage has not been fully elucidated. Researches have proved that lncRNA plays a major function in chemical-induced liver toxicity and can be used as a biomarker and therapeutic target for liver injury. In order to verify that lncRNA also participates in DMF-induced liver damage, we treated HL-7702 cells with 75 or 150 mM DMF, and obtained lncRNA expression profiles through high-throughput sequencing. Among the differentially expressed lncRNAs, lncRNA SNHG12 was proved to be significantly downregulated in DMF-treated HL-7702 cells and participate in DMF-mediated apoptosis, even under long-term low-dose DMF exposure (5–10 mM, 8 weeks). In addition, according to bioinformatics analysis, miR-218-5p is expected to be a potential target of SNHG12, which was verified by the dual luciferase reporter assay in HEK293FT cells. MiR-218-5p mimic can induce apoptosis in HL-7702 cells. Among the predicted targets of miR-218-5p, protein kinase C epsilon (PRKCE) was reported to be involved in apoptosis, and was indeed downregulated by miR-218-5p mimic in our study. Further experiments showed that changes of the expression of SNHG12 can affect the expression of PRKCE. In the epidemiological study of occupational population, we also found that SNHG12 was downregulated in the serum exosomes of workers exposed to DMF. These results indicated that SNHG12 can mediate DMF-induced apoptosis of HL-7702 cells through miR-218-5p/PRKCE pathway.

PKCe is highly expressed in mast cells and plays a fundamental role in the antigen-triggered activation of the allergic reaction. Although its regulation by diacylglycerols has been described, its regulation by acidic phospholipids and how this regulation leads to the control of downstream vesicle secretion is barely known. Here, we used structural and evolutionary studies to find the molecular mechanism that explains the selectivity of the C1B domain of PKCe by Phosphatidic Acid (PA). This resided in a collection of Arg residues that form a specific rim on the outer surface of the C1B domain, around the diacylglycerol binding cleft. In RBL-2H3 cells, this basic rim allowed the kinase to respond specifically to phosphatidic acid signals that induced its translocation to the plasma membrane and subsequent activation. Further experiments in cells that overexpress PKCe and a mutant of the PA binding site, showed that PA-dependent PKCe activation increased vesicle degranulation in RBL-2H3 cells, and this correlated with increased SNAP23 phosphorylation. Over-expression of PKCe in these cells also induced an increase in the number of docked vesicles containing SNAP23, when stimulated with PA. This accumulation could be attributed to the stabilizing effect of phosphorylation on the formation of the SNARE complex, which ultimately led to increased release of content in the presence of Ca2+ during the fusion process. Therefore, these findings reinforce the importance of PA signaling in the activation of PKCe, which could be an important target to inhibit the exacerbated responses of these cells in the allergic reaction.

Cyclase-associated protein 2 (CAP2) has been addressed as a candidate biomarker in various cancer types. Previously, we have shown that CAP2 is expressed during multi-step hepatocarcinogenesis; however, its underlying mechanisms in liver cancer cells are not fully elucidated yet. Here, we demonstrated that endoplasmic reticulum (ER) stress induced CAP2 expression, and which promoted migration and invasion of liver cancer cells. We also found that the ER stress-induced CAP2 expression is mediated through activation of protein kinase C epsilon (PKCe) and the promotor binding of activating transcription factor 2 (ATF2). In addition, we further demonstrated that CAP2 expression promoted epithelial-mesenchymal transition (EMT) through activation of Rac1 and ERK. In conclusion, we suggest that ER stress induces CAP2 expression promoting EMT in liver cancers cells. Our results shed light on the novel functions of CAP2 in the metastatic process of liver cancer cells.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

The presence of tight junction protein zonula occludens 2 (ZO-2) at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed whether the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knockdown Madin–Darby canine kidney cells, nuclear TEAD was diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation, and pull-down assays, TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated, and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals induces no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKCδ and ε and provide a novel role of ZO-2 as a nuclear translocator of TEAD.

Key points We have previously shown that carotid body stimulation by lysophosphatidic acid elicits a reflex stimulation of vagal efferent activity sufficient to cause bronchoconstriction in asthmatic rats. Here, we show that pathophysiological concentrations of asthma-associated prototypical Th2 cytokines also stimulate the carotid bodies. Stimulation of the carotid bodies by these asthmakines involves a PKCe-transient receptor potential vanilloid 1 (TRPV1) signalling mechanism likely dependent on TRPV1 S502 and T704 phosphorylation sites. As the carotid bodies' oxygen sensitivity is independent of PKCe-TRPV1 signalling, systemic blockade of PKCe may provide a novel therapeutic target to reduce allergen-induced asthmatic bronchoconstriction. Consistent with the therapeutic potential of blocking the PKCe-TRPV1 pathway, systemic delivery of a PKCe-blocking peptide suppresses asthmatic respiratory distress in response to allergen and reduces airway hyperresponsiveness to bradykinin. Abstract The autonomic nervous system orchestrates organ-specific, systemic and behavioural responses to inflammation. Recently, we demonstrated a vital role for lysophosphatidic acid in stimulating the primary autonomic oxygen chemoreceptors, the carotid bodies, in parasympathetic-mediated asthmatic airway hyperresponsiveness. However, the cacophony of stimulatory factors and cellular mechanisms of carotid body activation are unknown. Therefore, we set out to determine the intracellular signalling involved in carotid body-mediated sensing of asthmatic blood-borne inflammatory mediators. We employed a range of in vitro and rat in situ preparations, site-directed mutagenesis, patch-clamp, nerve recordings and pharmacological inhibition to assess cellular signalling. We show that the carotid bodies are also sensitive to asthma-associated prototypical Th2 cytokines which elicit sensory nerve excitation. This provides additional asthmatic ligands contributing to the previously established reflex arc resulting in efferent vagal activity and asthmatic bronchoconstriction. This novel sensing role for the carotid body is mediated by a PKCe-dependent stimulation of transient receptor potential vanilloid 1 (TRPV1), likely via TRPV1 phosphorylation at sites T704 and S502. Importantly, carotid body oxygen sensing was unaffected by blocking either PKCe or TRPV1. Further, we demonstrate that systemic PKCe blockade reduces asthmatic respiratory distress in response to allergen and airway hyperresponsiveness. These discoveries support an inflammation-dependent, oxygen-independent function for the carotid body and suggest that targeting PKCe provides a novel therapeutic option to abate allergic airway disease without altering life-saving autonomic hypoxic reflexes.

Doxorubicin (Dox)-induced cardiotoxicity (DIC) seriously threatens the health of related patients. Studies have confirmed that 14-3-3γ and protein kinase C epsilon (PKCε) are the endogenous protective proteins. Puerarin (Pue) is a bioactive ingredient isolated from the root of Pueraria lobata. It possesses many pharmacological properties, which have been widely used in treating and adjuvant therapy of cardiovascular diseases. In the study, we intended to explore the effects and mechanism of Pue pretreatment to protect the myocardium against DIC injury. Adult mice and H9c2 cells were pretreated with Pue, and the injury model was made with Dox. Results showed that Pue pretreatment alleviated DIC injury, as revealed by increased cell viability, decreased LDH activity and apoptosis, inhibited excess oxidative stress, maintained mitochondrial function and energy metabolism, and improved myocardial function. Furthermore, Pue pretreatment upregulated 14-3-3γ expression, interacted with PKCε, phosphorylated and impelled migration to mitochondria, activated adaptive autophagy, and protected the myocardium. However, pAD/14-3-3γ-shRNA or εV1-2 (a PKCε activity inhibitor) or 3-methyladenine (an autophagy inhibitor) could weaken the above effects of Pue pretreatment. Together, Pue pretreatment could activate adaptive autophagy by the 14-3-3γ/PKCε pathway and protect the myocardium against DIC injury.

Hypobaric hypoxia is a condition that occurs at high altitudes (>2500 m) where the partial pressure of gases, particularly oxygen (PO2), decreases. This condition triggers several physiological and molecular responses. One of the principal responses is pulmonary vascular contraction, which seeks to optimize gas exchange under this condition, known as hypoxic pulmonary vasoconstriction (HPV); however, when this physiological response is exacerbated, it contributes to the development of high-altitude pulmonary hypertension (HAPH). Increased levels of zinc (Zn2+) and oxidative stress (known as the “ROS hypothesis”) have been demonstrated in the vasoconstriction process. Therefore, the aim of this review is to determine the relationship between molecular pathways associated with altered Zn2+ levels and oxidative stress in HPV in hypobaric hypoxic conditions. The results indicate an increased level of Zn2+, which is related to increasing mitochondrial ROS (mtROS), alterations in nitric oxide (NO), metallothionein (MT), zinc-regulated, iron-regulated transporter-like protein (ZIP), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced protein kinase C epsilon (PKCε) activation in the development of HPV. In conclusion, there is an association between elevated Zn2+ levels and oxidative stress in HPV under different models of hypoxia, which contribute to understanding the molecular mechanism involved in HPV to prevent the development of HAPH.

Most individuals diagnosed with alcohol use disorders smoke cigarettes. Large concentrations of malondialdehyde and acetaldehyde are found in lungs co-exposed to cigarette smoke and alcohol. Aldehydes directly injure lungs and form aldehyde protein adducts, impacting epithelial functions. Recently, 2-(3-Amino-6-chloroquinolin-2-yl)propan-2-ol (ADX-102) was developed as an aldehyde-trapping drug. We hypothesized that aldehyde-trapping compounds are protective against lung injury derived from cigarette smoke and alcohol co-exposure. To test this hypothesis, we pretreated mouse ciliated tracheal epithelial cells with 0–100 µM of ADX-102 followed by co-exposure to 5% cigarette smoke extract and 50 mM of ethanol. Pretreatment with ADX-102 dose-dependently protected against smoke and alcohol induced cilia-slowing, decreases in bronchial epithelial cell wound repair, decreases in epithelial monolayer resistance, and the formation of MAA adducts. ADX-102 concentrations up to 100 µM showed no cellular toxicity. As protein kinase C (PKC) activation is a known mechanism for slowing cilia and wound repair, we examined the effects of ADX-102 on smoke and alcohol induced PKC epsilon activity. ADX-102 prevented early (3 h) activation and late (24 h) autodownregulation of PKC epsilon in response to smoke and alcohol. These data suggest that reactive aldehydes generated from cigarette smoke and alcohol metabolism may be potential targets for therapeutic intervention to reduce lung injury.

Background: Alzheimer’s disease is one of the neurodegenerative disorders typified by the aggregate of Aβ and phosphorylated tau protein. Oxidative stress and neuroinflammation, because of Aβ peptides, are strongly involved in the pathophysiology of AD. Linagliptin shows neuroprotective properties against AD pathological processes through alleviation of neural inflammation and AMPK activation. Methods: We assessed the benefits of linagliptin pretreatment (at 10, 20, and 50 nM concentrations), against Aβ1-42 toxicity (20 μM) in SH-SY5Y cells. The concentrations of secreted cytokines, such as TNF-α, IL-6, and IL-1β, and signaling proteins, including pCREB, Wnt1, and PKCε, were quantified by ELISA. Results: We observed that Aβ led to cellular inflammation, which was assessed by measuring inflammatory cytokines (TNF-α, IL-1β, and IL-6). Moreover, Aβ1-42 treatment impaired pCREB, PKCε, and Wnt1 signaling in human SH-SY5Y neuroblastoma cells. Addition of Linagliptin significantly reduced IL-6 levels in the lysates of cells, treated with Aβ1-42. Furthermore, linagliptin prevented the downregulation of Wnt1 in Aβ1-42-treated cells exposed. Conclusion: The current findings reveal that linagliptin alleviates Aβ1-42-induced inflammation in SH-SY5Y cells, probably through the suppression of IL-6 release, and some of its benefits are mediated through the activation of the Wnt1 signaling pathway.

Background Our previous study has demonstrated that morphine post-conditioning (MpostC) protects cardiomyocytes from ischemia/reperfusion (I/R) injury partly through activating protein kinase-epsilon (PKCε) signaling pathway and subsequently inhibiting mitochondrial permeability transition pore (mPTP) opening. Aim In this study, we aim to investigate the relationship between long non-coding RNA TINCR and PKCε in cardiomyocytes under MpostC-treated I/R injury. Design The myocardial I/R rat model was established by the ligation of lower anterior descending coronary artery for 45 min followed by the reperfusion for 1 h, and MpostC was performed before the reperfusion. Method H/R and MpostC were performed in the rat cardiomyocyte cell line (H9C2), and the Cytochrome-c release in cytosol and mPTP opening were determined. Cell viability was detected by using Cell Counting Kit-8, and cell apoptosis was determined by using flow cytometry or TUNEL assay. Results The results indicated that MpostC restored the expression of TINCR in I/R rat myocardial tissues. In cardiomyocytes, the therapeutic effect of MpostC, including reduced mPTP opening, reduced Cytochrome-c expression, increased cell viability and reduced cell apoptosis, was dramatically negated by interfering TINCR. The expression of fibroblast growth factor 1 (FGF1), a protein that activates PKCε signaling pathway, was positively correlated with TINCR. The RNA immunoprecipitation and RNA pull-down assay further confirmed the binding between FGF1 and TINCR. Furthermore, TINCR was demonstrated to inhibit the degradation and ubiquitination of FGF1 in cardiomyocytes using the cycloheximide experiment and the ubiquitination assay. The TINCR/FGF1/PKCε axis was revealed to mediate the protective effect of MpostC against hypoxia/reoxygenation injury both in vitro and in vivo. Conclusion In conclusion, our findings demonstrated that MpostC-induced up-regulation of TINCR protects cardiomyocytes from I/R injury via inhibiting degradation and ubiquitination of FGF1, and subsequently activating PKCε signaling pathway, which provides a novel insight in the mechanism of TINCR and PKCε during MpostC treatment of I/R injury.

The failure of insulin to suppress glucose production by the liver is a key aspect of the insulin resistance seen in type 2 diabetes. Lipid-activated protein kinase C epsilon has long been identified as an important mediator of diet-induced glucose intolerance and hepatic insulin resistance and the current view emphasizes a mechanism involving phosphorylation of the insulin receptor by the kinase to inhibit downstream insulin action. However, the significance of this direct effect in the liver has now been challenged by tissue-specific deletion of PKCε, which demonstrated a more prominent role for the kinase in adipose tissue to promote glucose intolerance. New insights regarding the role of PKCε therefore contribute to the understanding of indirect effects on hepatic glucose metabolism.

Human embryonic stem cells (hESCs) have unlimited proliferation capacity and can differentiate into most types of somatic cells. We previously described that the overexpression of FLI1 as well as the activation of protein kinase C (FLI1-PKC) could rapidly and efficiently differentiate hESCs into endothelial cells (ECs). However, the relationship between vascular endothelial growth factor (VEGF) and PKC in hESC-EC differentiation is debated, and the roles of different PKC isoforms in hESC-EC differentiation remain unknown. In this study, after 2 days of induction, we found that the overexpression of FLI1 and the addition of VEGF-A to hESCs (FLI1-VEGF) could generate 19.6% ± 5.4% ECs. The induction efficiency of ECs was reduced from 72.3% ± 7.6% to 37.9% ± 4.9% following the addition VEGF-A siRNA to the FLI1-PKC system. However, the induction of ECs was nearly completely abrogated following the addition of the pan-PKC inhibitor to the FLI1-VEGF, FLI1-PKC, or other systems. The above results suggested that VEGF can partially replace PKC, but PKC plays a more critical role downstream of VEGF during hESC-EC induction. To further investigate which PKC isoform was mainly involved in converting hESCs to ECs, siRNAs were used to knock down nine PKC isoforms in the FLI1-PKC system. Only the knockdown of PKC-ɛ or PKC-η significantly decreased the induction efficiency of ECs to 51.1% ± 5.8% or 50.3% ± 5.1%, respectively. PKC-ɛ/η siRNA could suppress EC induction in other differentiation systems. Moreover, RNA-seq and quantitative polymerase chain reaction analysis also showed that only the expression of PKC-ɛ and PKC-η was robustly upregulated during hESC-EC induction. In summary, our results suggested that VEGF promoted the differentiation of ECs derived from hESCs, which mainly depended on PKC, specifically the PKC-ɛ and PKC-η pathways.

Irritable bowel syndrome (IBS) is a clinical disease mainly characterized as a syndrome of abdominal pain and discomfort, which frequently occurs in humans aged 20-50. Abdomen massage is of great medical significance for the health of the human body, including promoting intestinal peristalsis, relieving constipation, and facilitating weight loss. However, its potential benefits in alleviating IBS and the underlying mechanisms remain elusive.In this study, we established an IBS model in rats to evaluate the effects of abdomen massage. Forty male Sprague Dawley (SD) rats were randomly assigned into 4 groups: the normal (control) group, IBS group, abdominal massage group, and abdominal massage + ketotifen treatment group (We found that compared with the control group, the mast cells in the IBS group were significantly increased and the increased MC was partially decreased by an abdominal massage with or without ketotifen treatment. We also found that TRPV1 was upregulated in the IBS group. Abdominal massage with or without ketotifen treatment could attenuate the upregulation of TRPV1 in IBS. Mechanically, results of IHC and western Blot suggested that abdominal massage reduces the sensitivity of IBS by regulating the trypase-PAR2-PKCOverall, our results suggested that abdominal massage produces a beneficial effect in improving the symptoms of IBS through reducing mast cell recruitment and attenuating the trypase-PAR2-PKC

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new member of the Betacoronaviridae family, responsible for the recent pandemic outbreak of COVID-19. To start exploring the molecular events that follow host cell infection, we queried VirusCircBase and identified a circular RNA (circRNA) predicted to be synthesized by SARS-CoV-2, circ_3205, which we used to probe: (i) a training cohort comprised of two pools of cells from three nasopharyngeal swabs of SARS-CoV-2 infected (positive) or uninfected (negative, UCs) individuals; (ii) a validation cohort made up of 12 positive and 3 negative samples. The expression of circRNAs, miRNAs and miRNA targets was assayed through real-time PCR. CircRNA–miRNA interactions were predicted by TarpMiR, Analysis of Common Targets for circular RNAs (ACT), and STarMir tools. Enrichment of the biological processes and the list of predicted miRNA targets were retrieved from DIANA miRPath v3.0. Our results showed that the predicted SARS-CoV-2 circ_3205 was expressed only in positive samples and its amount positively correlated with that of SARS-CoV-2 Spike (S) mRNA and the viral load (r values = 0.80952 and 0.84867, Spearman’s correlation test, respectively). Human (hsa) miR-298 was predicted to interact with circ_3205 by all three predictive tools. KCNMB4 and PRKCE were predicted as hsa-miR-298 targets. Interestingly, the function of both is correlated with blood coagulation and immune response. KCNMB4 and PRKCE mRNAs were upregulated in positive samples as compared to UCs (6 and 8.1-fold, p values = 0.049 and 0.02, Student’s t test, respectively) and their expression positively correlated with that of circ_3205 (r values = 0.6 and 0.25, Spearman’s correlation test, respectively). We propose that our results convincingly suggest that circ_3205 is a circRNA synthesized by SARS-CoV-2 upon host cell infection and that it may behave as a competitive endogenous RNA (ceRNA), sponging hsa-miR-298 and contributing to the upregulation of KCNMB4 and PRKCE mRNAs.

Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7β-hydroxycholesterol (7βOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7βOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7βOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7βOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7βOH as a candidate marker for evaluating cardiovascular risk factors in obesity.

Hypericum perforatum L., commonly known as St. John's Wort (SJW), represents one of the best-known and most thoroughly researched medicinal plant species. The ethnobotanical usage and bioactivities related to H. perforatum include treatment of skin diseases, wounds and burns, gastrointestinal problems, urogenital diseases and psychiatric disorders, particularly depression. In the last decade, many studies focused on the bioactive constituents responsible for the antihyperglycemic and antidiabetic activity of SJW extracts. However, the mechanism by which H. perforatum extract exhibits these properties is still unclear. Hence, the current study was designed to gain insight into the underlying biochemical and molecular mechanisms by which wildly growing H. perforatum exerts its antihyperglycemic and antidiabetic activities.Plant material of H. perforatum was harvested from a natural population in the Republic of North Macedonia during full flowering season. Methanol (80% v/v) was used to extract bioactive components from HH powder. The dissolved HH dry extract (in 0.3% CMC) was given daily as a single treatment (200 mg/kg bw) during 14 days both in healthy and streptozotocin-induced diabetic rats. As a positive control, we applied glibenclamide. The activity of key enzymes involved in carbohydrate methabolisam in the liver were assessed, along with substrate concentration, as well as AMPK mRNA levels, PKCε concentration, plasma insulin level and pancreatic PARP activity.Compared to diabetic rats, treatment of diabetic rats with HH extract resulted with decreased activity of hepatic enzymes glucose-6-phospatase and fructose-1,6-bisphosphatase, increased liver glycogen and glucose-6-phosphate content, which resulted with reduced blood glucose concentration up to normoglycaemia. Non-significant changes were observed in the activity of hexokinase, glycogen phosphorylase and glucose-6-phospahte dehydrogenase. HH-treatment also caused an increase in plasma insulin concentration and increase in pancreatic PARP activity. Finally, HH treatment of diabetic rats showed significant increase in AMPK expression and decrease of PKCε concentration.We present in vivo evidence that HH- extract exert insulinotropic effects and regulate endogenous glucose production mostly by suppressing liver gluconeogenesis. The HH-treatment did not effected glycogenolysys and glycolysis. Finally, we confirm the antihyperglycemic and antidiabetic effect of HH-extract and the mechanism of this effect involves amelioration of AMPK and PKCε changes in the liver.

sn-1,2-diacylglycerol (sn-1,2-DAG)-mediated activation of protein kinase Cε (PKCε) is a key pathway that is responsible for obesity-related lipid metabolism disorders, which induces hepatic insulin resistance and type 2 diabetes. No small molecules have been previously reported to ameliorate these diseases through this pathway. Here, we screened and identified the phytochemical atractylenolide II (AT II) that reduces the hepatic sn-1,2-DAG levels, deactivates PKCε activity, and improves obesity-induced hyperlipidemia, hepatosteatosis, and insulin resistance. Furthermore, using the ABPP strategy, the diacylglycerol kinase family member DGKQ was identified as a direct target of AT II. AT II may act on a novel drug-binding pocket in the CRD and PH domains of DGKQ to thereby allosterically regulate its kinase activity. Moreover, AT II also increases weight loss by activating DGKQ-AMPK-PGC1α-UCP-1 signaling in adipose tissue. These findings suggest that AT II is a promising lead compound to improve obesity-induced insulin resistance.

Lyciumamide A (LyA), a dimer of phenolic amide isolated from the fruits of Lycium barbarum, has been confirmed to possess potent antioxidant activity. This study was aimed to investigate the neuroprotection and molecular mechanisms of LyA against cerebral ischemia/reperfusion (I/R) injury via improving antioxidant activity. The model of middle cerebral artery occlusion (MCAO) and SH-SY5Y cells induced by oxygen and glucose deprivation (OGD) were adopted to verify the neuroprotective effects and the potential pharmacology mechanisms of LyA in vivo and in vitro. In MCAO model, treatment with LyA significantly improved neurologic score, reduced infarct volume, and relieved oxidative stress injury at 48 h after reperfusion. Meanwhile, LyA markedly increased the transcription Nrf2 and HO-1 expressions in the ischemic cerebral cortex. In vitro results showed that LyA protected differentiated SH-SY5Y cells against OGD-induced injury. LyA significantly decreased the expression of caspase-3 and the Bax/Bcl-2 ratio. But knockdown of Nrf2 or HO-1 attenuated the protective effect of LyA. Similarly, knockdown of protein kinase Ce (PKCe) inhibited LyA-induced Nrf2/HO-1 activation, and abated its protective effects. In conclusion, this study firstly demonstrated that LyA protects against cerebral I/R injury, ameliorates oxidative damage and neuronal apoptosis, partly via activation of PKCe/Nrf2/HO-1 pathway.

Studies have confirmed that the heart is the main target organ of lipopolysaccharide (LPS) attacks, and 14-3-3γ and protein kinase C epsilon (PKCε) are the endogenous protective proteins. Puerarin (Pue) is the major bioactive ingredient isolated from the root of Pueraria lobata. It possesses many pharmacological properties, which has been widely used in the treatment and adjuvant therapy of cardio- and cerebrovascular diseases and cancer, etc. The study intended to explore the effects and mechanism of Pue pretreatment to protect myocardium against LPS injury. Adult mice and primary cultured neonatal rat cardiomyocytes were pretreated with Pue, and the injury model was made with LPS. Results showed that Pue pretreatment alleviated LPS-induced injury, as demonstrated by increased cell viability, decreased LDH activity and apoptosis, inhibited excess oxidative stress and the inflammatory cytokine release, and maintained mitochondrial function. Furthermore, Pue pretreatment upregulated 14-3-3γ expression, interacted with PKCε, which was phosphorylated and impelled migration to mitochondria, and then activated adaptive autophagy and protected the myocardium. However, pAD/14-3-3γ-shRNA or 3-MA (an autophagy inhibitor) could weaken the above effects of Pue pretreatment. Together, Pue pretreatment could activate adaptive autophagy by the 14-3-3γ/PKCε pathway and protect the myocardium against LPS injury.

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

The PKCε-regulated genome protective pathway provides transformed cells a failsafe to successfully complete mitosis. Despite the necessary role for Aurora B in this programme, it is unclear whether its requirement is sufficient or if other PKCε cell cycle targets are involved. To address this, we developed a trapping strategy using UV-photocrosslinkable amino acids encoded in the PKCε kinase domain. The validation of the mRNA binding protein SERBP1 as a PKCε substrate revealed a series of mitotic events controlled by the catalytic form of PKCε. PKCε represses protein translation, altering SERBP1 binding to the 40 S ribosomal subunit and promoting the assembly of ribonucleoprotein granules containing SERBP1, termed M-bodies. Independent of Aurora B, SERBP1 is shown to be necessary for chromosome segregation and successful cell division, correlating with M-body formation. This requirement for SERBP1 demonstrates that Aurora B acts in concert with translational regulation in the PKCε-controlled pathway exerting genome protection., PKCε is known to exert a role in genome protection by directly phosphorylating and switching the specificity of Aurora B. Here the authors identify SERBP1 as a parallel mitotic PKCε substrate controlling translation and ensuring the integrity of chromosome segregation and successful cell division.

Protein kinase C (PKC)-ε is required for membrane addition during IgG-mediated phagocytosis, but its role in this process is ill defined. Here, we performed high-resolution imaging, which reveals that PKC-ε exits the Golgi and enters phagosomes on vesicles that then fuse. TNF and PKC-ε colocalize at the Golgi and on vesicles that enter the phagosome. Loss of PKC-ε and TNF delivery upon nocodazole treatment confirmed vesicular transport on microtubules. That TNF+ vesicles were not delivered in macrophages from PKC-ε null mice, or upon dissociation of the Golgi-associated pool of PKC-ε, implies that Golgi-tethered PKC-ε is a driver of Golgi-to-phagosome trafficking. Finally, we established that the regulatory domain of PKC-ε is sufficient for delivery of TNF+ vesicles to the phagosome. These studies reveal a novel role for PKC-ε in focal exocytosis – its regulatory domain drives Golgi-derived vesicles to the phagosome, whereas catalytic activity is required for their fusion. This is one of the first examples of a PKC requirement for vesicular trafficking and describes a novel function for a PKC regulatory domain.This article has an associated First Person interview with the first author of the paper.

A rat hyperalgesia model was induced using an intraplantar injection of Freund's complete adjuvant (FCA) or an intrathecal injection of IL-6. Mechanical allodynia was evaluated using von Frey filament tests after intrathecal injections of T-5224 (c-Fos/AP-1 inhibitor), minocycline (Mino, a specific microglia inhibitor), L-2-aminoadipic acid (LAA, an astroglial toxin), PKCT-5224, Mino, and LAA attenuated FCA- or IL-6-mediated inflammatory pain, with a decrease in c-Fos, GFAP, Iba-1, PKCSTAT3 phosphorylation at Ser 727 and the interaction with PKC

Mitochondria, abundant organelles in high energy demand cells such as cardiomyocytes, can determine cell death or survival by regulating the opening of mitochondrial permeability transition pore, mPTP. We addressed the hypothesis that the growth factor FGF2, known to reside in intracellular locations, can directly influence mitochondrial susceptibility to mPTP opening. Rat cardiac subsarcolemmal (SSM) or interfibrillar (IFM) mitochondrial suspensions exposed directly to rat 18 kDa low molecular weight (Lo-) FGF2 isoform displayed increased resistance to calcium overload-induced mPTP, measured spectrophotometrically as “swelling”, or as cytochrome c release from mitochondria. Inhibition of mitochondrial protein kinase C epsilon abrogated direct Lo-FGF2 mito-protection. Exposure to the rat 23 kDa high molecular weight (Hi) FGF2 isoform promoted cytochrome c release from SSM and IFM under nonstressed conditions. The effect of Hi-FGF2 was prevented by mPTP inhibitors, pre-exposure to Lo-FGF2, and okadaic acid, a serine/threonine phosphatase inhibitor. Western blotting and immunoelectron microscopy pointed to the presence of immunoreactive FGFR1 in cardiac mitochondria in situ. The direct mito-protective effect of Lo-FGF2, as well as the deleterious effect of Hi-FGF2, were prevented by FGFR1 inhibitors and FGFR1 neutralizing antibodies. We propose that intracellular FGF2 isoforms can modulate mPTP opening by interacting with mito-FGFR1 and relaying isoform-specific intramitochondrial signal transduction.