PKCβ Activation Research Articles

Y4 RNA fragments from cardiosphere-derived cells ameliorate diabetic myocardial ischemia‒reperfusion injury by inhibiting protein kinase C β-mediated macrophage polarization

The specific pathophysiological pathways through which diabetes exacerbates myocardial ischemia/reperfusion (I/R) injury remain unclear; however, dysregulation of immune and inflammatory cells, potentially driven by abnormalities in their number and function due to diabetes, may play a significant role. In the present investigation, we simulated myocardial I/R injury by inducing ischemia through ligation of the left anterior descending coronary artery in mice for 40 min, followed by reperfusion for 24 h. Previous studies have indicated that protein kinase Cβ (PKCβ) is upregulated under hyperglycemic conditions and is implicated in the development of various diabetic complications. The Y4 RNA fragment is identified as the predominant small RNA component present in the extracellular vesicles of cardio sphere-derived cells (CDCs), exhibiting notable anti-inflammatory properties in the contexts of myocardial infarction and cardiac hypertrophy. Our investigation revealed that the administration of Y4 RNA into the ventricular cavity of db/db mice following myocardial I/R injury markedly enhanced cardiac function. Furthermore, Y4 RNA was observed to facilitate M2 macrophage polarization and interleukin-10 secretion through the suppression of PKCβ activation. The mechanism by which Y4 RNA affects PKCβ by regulating macrophage activation within the inflammatory environment involves the inhibition of ERK1/2 phosphorylation In our study, the role of PKCβ in regulating macrophage polarization during myocardial I/R injury was investigated through the use of PKCβ knockout mice. Our findings indicate that PKCβ plays a crucial role in modulating the inflammatory response associated with macrophage activation in db/db mice experiencing myocardial I/R, with a notable exacerbation of this response observed upon significant upregulation of PKCβ expression. In vitro studies further elucidated the protective mechanism by which Y4 RNA modulates the PKCβ/ERK1/2 signaling pathway to induce M2 macrophage activation. Overall, our findings suggest that Y4 RNA plays an anti-inflammatory role in diabetic I/R injury, suggesting a novel therapeutic approach for managing myocardial I/R injury in diabetic individuals.

Role of Protein Kinase C in Metabolic Regulation of Coronary Endothelial Small Conductance Calcium-Activated Potassium Channels.

Small conductance calcium-activated potassium (SK) channels are largely responsible for endothelium-dependent coronary arteriolar relaxation. Endothelial SK channels are downregulated by the reduced form of nicotinamide adenine dinucleotide (NADH), which is increased in the setting of diabetes, yet the mechanisms of these changes are unclear. PKC (protein kinase C) is an important mediator of diabetes-induced coronary endothelial dysfunction. Thus, we aimed to determine whether NADH signaling downregulates endothelial SK channel function via PKC. SK channel currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence/absence of NADH, PKC activator phorbol 12-myristate 13-acetate, PKC inhibitors, or endothelial PKCα/PKCβ knockdown by using small interfering RNA. Human coronary arteriolar reactivity in response to the selective SK activator NS309 was measured by vessel myography in the presence of NADH and PKCβ inhibitor LY333531. NADH (30-300 μmol/L) or PKC activator phorbol 12-myristate 13-acetate (30-300 nmol/L) reduced endothelial SK current density, whereas the selective PKCᵦ inhibitor LY333531 significantly reversed the NADH-induced SK channel inhibition. PKCβ small interfering RNA, but not PKCα small interfering RNA, significantly prevented the NADH- and phorbol 12-myristate 13-acetate-induced SK inhibition. Incubation of human coronary artery endothelial cells with NADH significantly increased endothelial PKC activity and PKCβ expression and activation. Treating vessels with NADH decreased coronary arteriolar relaxation in response to the selective SK activator NS309, and this inhibitive effect was blocked by coadministration with PKCβ inhibitor LY333531. NADH-induced inhibition of endothelial SK channel function is mediated via PKCβ. These findings may provide insight into novel therapeutic strategies to preserve coronary microvascular function in patients with metabolic syndrome and coronary disease.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-β activation. Activated PKC-β induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

The store-operated Ca2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1β, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1β lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1β is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCβ activation. Our results indicate that Orai1α interacts with PKCβ2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.

ZiBuPiYin recipe ameliorates diabetes-associated cognitive decline by improving neuronal mitochondrial function in chronic psychologically stressed zucker diabetic fatty rats

Ethnopharmacological relevanceZibu Piyin Recipe (ZBPYR) is a traditional Chinese medicine compound composed of 12 kinds of Chinese herbal medicines including red ginseng and yam. Long-term basic and clinical applications have proved that ZBPYR can prevent and treat cognitive dysfunction. Previous studies showed that chronic psychological stress can increase the risk of type 2 diabetes mellitus (T2DM), and lead to cognitive decline. Mitochondrial dysfunction plays a key role in chronic psychological stress-induced diabetes mellitus. While the mechanism of mitochondrial dysfunction and insulin resistance in diabetes-associated cognitive decline (DACD) is unclear. Aim of the studyOur previous research found that a ZiBuPiYin recipe (ZBPYR) has significant pharmacological effects against DACD. The present study investigated changes in mitochondrial dysfunction in the brain and the mechanism of insulin resistance and mitochondrial damage to explore the relationship between neuronal mitochondrial dysfunction and insulin resistance in chronic psychologically stressed DACD rats. Materials and methodsZucker diabetic fatty (ZDF) rats with spontaneous T2DM and rats with diabetic cognitive impairment that was induced by chronic psychological stress were used in in vivo experiments. PC12 cells that were damaged by rotenone were used for the in vitro experiment. ResultsThe findings indicated that the number of mitochondria decreased, morphology and membrane potential were damaged, and reactive oxygen species increased in the cortex and hippocampus in psychologically stressed DACD rats. Protein kinase Cβ2 (PKCβ2) activation and insulin resistance were markedly induced by chronic psychological stress, together with decreases in peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial fusion protein 2 (Mfn2). Furthermore, ZBPYR exerted protective effects both in in vivo and in vitro. ConclusionMitochondrial damage and insulin resistance were observed in the brain in chronic psychologically stressed DACD rats. The ZBPYR significantly improved brain mitochondrial damage and insulin resistance in chronic psychologically stressed DACD rats. These results provide novel insights for the development of ZBPYR as a traditional Chinese medicine for the treatment of chronic psychological stress and DACD.

Abstract P125: Long-term Exposure Of Angiotensin II Enhanced Vascular Contraction Via Activation Of Protein Kinase C Beta

Protein kinase C (PKC) β is linked to lymphoma and diabetes complications including diabetic retinopathy, nephropathy and neuropathy. Also, recent studies revealed that PKCβ activation also accelerates atherosclerotic plaque formation in diabetes. These findings strongly indicate that PKCβ is a potential target for the treatment of diabetic vascular complications. Although PKCβ is associated with diabetic vascular dysfunction, role of PKCβ in hypertension remains unclear. Since hypertension is one of most important risk factor for vascular diseases including atherosclerosis, we here examined whether PKCβ is involved in angiotensin II-induced vascular dysfunction. Treatment with angiotensin II (100 nM) for 30 min strongly increased phosphorylation at the PKCβ activation site at S660 in aorta from Wistar rat (n=4). Treatment of rat aorta with angiotensin II (100 nM) for 24 hr increased contractile responses to serotonin (5-HT, Maximum contraction, control vs. Angiotensin II, 92.2±20.0 vs 142.1±20.5%, P<0.05, n=6) or endothelin-1 (ET-1, 157.4±30.6 vs 214.1±24.3%, P<0.05, n=8). In contrast, angiotensin II did not affect vascular contractile response to noradrenaline (n=4). The enhanced contraction induced by angiotensin II was blocked by CGP53353 (1 μM), a PKCβ inhibitor (5-HT, Angiotensin II vs. CGP53353+Angiotensin II,197.8±27.8 vs. 167.4±17.6%, P<0.05, n=4-5). Pretreatment with LY333531 (1 μM), another PKCβ inhibitor, also prevented angiotensin II-induced vascular dysfunction (P<0.05, n=3-4). Next, we generated PKCβ knockout rat using rGONAD. Consistent with the results using PKCβ inhibitors, angiotensin II-induced enhanced contractile responses to 5-HT (122.6±15.6 vs 111.0±16.3%, n=4) and ET-1 (137.9±39.9 vs 139.2±30.1%, n=4) were dismissed in PKCβ knockout rat. We also confirmed that phorbol12-myristate13-acetate (0.05-5 μg/ml), a PKC activator, enhanced vascular contractile responses to 5-HT and ET-1 in dose-dependent manner (P<0.05, n=4). We conclude that long exposure of angiotensin II (24 hr) causes increase in vascular contractile responses to 5-HT and ET-1 and the augmented contraction are due to activation of PKCβ. Our result suggests that PKCβ might be associated with angiotensin II-related vascular dysfunction.

PKCβ Inhibition Promotes TXNIP Degradation to Ameliorate Pancreatic β-Cell Dysfunction

Introduction: Pancreatic β-cell dysfunction is largely regulated by TXNIP accumulation, we have previously disclosed the role of PKA in TXNIP degradation during β-cell dysfunction. However, whether other kinases (PKCs) still regulate TXNIP is unclear, which is beneficial to alleviate β-cell dysfunction. Methods: Thapsigargin (ER stress inducer) was used to induce β-cell dysfunction. PKC’s inhibitors were screened by Western blotting indicated by TXNIP. Also RT-qPCR and Co-immunoprecipitation were applied for evaluating the β-cell improvement ability of PKC’s inhibitors, and the insulin secretion ability was evaluated by glucose-stimulated insulin secretion assay. Results: PKC’s pan-inhibitor, Ro31-8220, decreased β-cell apoptosis and improved insulin secretion under ER stress or high glucose (HG) conditions. Further studies showed that Ro31-8220 reduced ER stress or HG-induced TXNIP levels. On the other side, PKCβ activation or overexpression could reverse the effect of Ro31-8220 on TXNIP. Also, PKCβ selective inhibitor, ruboxistaurin, induced TXNIP degradation as significantly as Ro31-8220 did. Conclusion: This study reveals the regulating mechanism of PKCβ inhibitor on TXNIP degradation to improve β-cell dysfunction. These data indicated PKCβ inhibitor is a promising agent for ameliorating β-cell dysfunction through TXNIP.

Activation of Coronary Arteriolar PKCβ2 Impairs Endothelial NO-Mediated Vasodilation: Role of JNK/Rho Kinase Signaling and Xanthine Oxidase Activation.

Protein kinase C (PKC) activation can evoke vasoconstriction and contribute to coronary disease. However, it is unclear whether PKC activation, without activating the contractile machinery, can lead to coronary arteriolar dysfunction. The vasoconstriction induced by the PKC activator phorbol 12,13-dibutyrate (PDBu) was examined in isolated porcine coronary arterioles. The PDBu-evoked vasoconstriction was sensitive to a broad-spectrum PKC inhibitor but not affected by inhibiting PKCβ2 or Rho kinase. After exposure of the vessels to a sub-vasomotor concentration of PDBu (1 nmol/L, 60 min), the endothelium-dependent nitric oxide (NO)-mediated dilations in response to serotonin and adenosine were compromised but the dilation induced by the NO donor sodium nitroprusside was unaltered. PDBu elevated superoxide production, which was blocked by the superoxide scavenger Tempol. The impaired NO-mediated vasodilations were reversed by Tempol or inhibition of PKCβ2, xanthine oxidase, c-Jun N-terminal kinase (JNK) and Rho kinase but were not affected by a hydrogen peroxide scavenger or inhibitors of NAD(P)H oxidase and p38 kinase. The PKCβ2 protein was detected in the arteriolar wall and co-localized with endothelial NO synthase. In conclusion, activation of PKCβ2 appears to compromise NO-mediated vasodilation via Rho kinase-mediated JNK signaling and superoxide production from xanthine oxidase, independent of the activation of the smooth muscle contractile machinery.

Abstract P378: Metabolic Dysregulation Of Endothelial Sk Channels Via Protein Kinase C

Introduction: Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, which predisposes to ischemic cardiovascular events. Small conductance calcium-activated-potassium (SK) channels are largely responsible for coronary arteriolar relaxation mediated by endothelium-dependent hyperpolarizing factors. Diabetic inactivation/inhibition of endothelial SK channels contributes to endothelial dysfunction. Endothelial dysfunction during diabetes (DM) is also associated with increases in metabolites NADH, and PKC. The pyridine nucleotide NADH has been recently found to inactivate endothelial SK channels. The overexpressed and activated PKC has been shown to play an important role in diabetes-induced endothelial dysfunction. However, it is undefined if PKC is involved in the metabolite NADH dysregulation of endothelial SK channels. Hypothesis: We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels. Methods: SK channels currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence or absence of NADH, and/or PKC activator PMA, PKC inhibitors or endothelial PKC α /PKCβ knock-down by using short interfering RNA. Results: NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p<0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC α inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p>0.05 vs. control (n=15), Fig. E). PKC α knock-down failed to affect NADH (n=13) and PMA (n=10) inhibition of endothelial SK currents (Fig. F). In contrast, PKCβ knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p>0.05, vs. control, Fig. G). Conclusions: The metabolite NADH dysregulation of endothelial SK channels was via PKCβ activation.

Selective inhibition of PKCβ2 improves Caveolin-3/eNOS signaling and attenuates lipopolysaccharide-induced injury by inhibiting autophagy in H9C2 cardiomyocytes.

Lipopolysaccharide (LPS)-induced autophagy is involved in sepsis-associated myocardial injury with increased PKCβ2 activation. We previously found hyperglycemia-induced PKCβ2 activation impaired the expression of caveolin-3 (Cav-3), the dominant isoform to form cardiomyocytes caveolae which modulate eNOS signaling to confer cardioprotection in diabetes. However, little is known about the roles of PKCβ2 in autophagy and Cav-3/eNOS signaling in cardiomyocytes during LPS exposure. We hypothesize LPS-induced PKCβ2 activation promotes autophagy and impairs Cav-3/eNOS signaling in LPS-treated cardiomyocytes. H9C2 cardiomyocytes were treated with LPS (10µg/mL) in the presence or absence of PKCβ2 inhibitor CGP53353 (CGP, 1µM) or autophagy inhibitor 3-methyladenine (3-MA, 10µM). LPS stimulation induced cytotoxicity overtime in H9C2 cardiomyocytes, accompanied with excessive PKCβ2 activation. Selective inhibition of PKCβ2 with CGP significantly reduced LPS-induced cytotoxicity and autophagy (measured by LC-3II, Beclin-1, p62 and autophagic flux). In addition, CGP significantly attenuated LPS-induced oxidative injury, and improved Cav-3 expression and eNOS activation, similar effects were shown by the treatment of autophagy inhibitor 3-MA. LPS-induced myocardial injury is associated with excessive PKCβ2 activation, which contributes to elevated autophagy and impaired Cav-3/eNOS signaling. Selective inhibition of PKCβ2 improves Cav-3/eNOS signaling and attenuates LPS-induced injury through inhibiting autophagy in H9C2 cardiomyocytes.

Anti-Malignant Ascites Effect of Total Diterpenoids from Euphorbiae Ebracteolatae Radix Is Attributable to Alterations of Aquaporins via Inhibiting PKC Activity in the Kidney.

This study evaluated the anti-ascites effect of total diterpenoids extracted from Euphorbiae ebracteolatae Radix (TDEE) on malignant ascitic mice and elucidated its underlying mechanism. TDEE was extracted by dichloromethane and subjected to column chromatography. The purity of six diterpenoids isolated from TDEE was determined to be 77.18% by HPLC. TDEE (3 and 0.6 g raw herbs/kg, p.o.) reduced ascites and increased urine output. Meanwhile, analysis of tumor cell viability, cycle and apoptosis indicated that TDEE had no antitumor activity. In addition, the expression levels of aquaporins (AQPs) and the membrane translocation levels of protein kinase C (PKC) α and PKCβ in kidney and cells were measured. TDEE reduced the levels of AQP1–4, and inhibited PKCβ expression in membrane fraction. Four main diterpenoids, except compound 2, reduced AQP1 level in human kidney-2 cells. Compounds 4 and 5 inhibited AQP2–4 expression in murine inner medullary collecting duct cells. The diterpenoid-induced inhibition of AQP1–4 expression was blocked by phorbol-12-myristate-13-acetate (PMA; agonist of PKC). The diterpenoids from TDEE are the main anti-ascites components. The anti-ascites effect of diterpenoids may be associated with alterations in AQPs in the kidneys to promote diuresis. The inhibition of AQP1–4 expression by TDEE is related to the inhibition of PKCβ activation.

Selective Inhibition of PKCβ2 Restores Ischemic Postconditioning-Mediated Cardioprotection by Modulating Autophagy in Diabetic Rats.

Diabetic hearts are more susceptible to myocardial ischemia/reperfusion (I/R) injury and less sensitive to ischemic postconditioning (IPostC), but the underlying mechanisms remain unclear. PKCβ2 is preferentially overactivated in diabetic myocardium, in which autophagy status is abnormal. This study determined whether hyperglycemia-induced PKCβ2 activation resulted in autophagy abnormality and compromised IPostC cardioprotection in diabetes. We found that diabetic rats showed higher cardiac PKCβ2 activation and lower autophagy than control at baseline. However, myocardial I/R further increased PKCβ2 activation and promoted autophagy status in diabetic rats. IPostC significantly attenuated postischemic infarct size and CK-MB, accompanied with decreased PKCβ2 activation and autophagy in control but not in diabetic rats. Pretreatment with CGP53353, a selective inhibitor of PKCβ2, attenuated myocardial I/R-induced infarction and autophagy and restored IPostC-mediated cardioprotection in diabetes. Similarly, CGP53353 could restore hypoxic postconditioning (HPostC) protection against hypoxia reoxygenation- (HR-) induced injury evidenced by decreased LDH release and JC-1 monomeric cells and increased cell viability. These beneficial effects of CGP53353 were reversed by autophagy inducer rapamycin, but could be mimicked by autophagy inhibitor 3-MA. It is concluded that selective inhibition of PKCβ2 could attenuate myocardial I/R injury and restore IPostC-mediated cardioprotection possibly through modulating autophagy in diabetes.

PKCβ and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats.

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCβ and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCβ and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCβ inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCβ-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCβ in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCβ activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCβ and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

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.

Rituximab and Obinutuzumab Induce Direct B-Cell Death Via B-Cell Receptor (BCR) Signaling, but Rituximab Elicits Stronger BCR-Derived Pro-Survival Signals Diminishing Apoptosis

The anti-CD20 monoclonal antibody (mAb) rituximab in combination with chemotherapy has improved outcomes for patients with CD20+ B-cell lymphoma. Obinutuzumab was developed as an anti-CD20 mAb with enhanced induction of direct B-cell death and antibody-dependent cellular cytotoxicity. In direct comparison to rituximab, improved outcomes with obinutuzumab have been observed for chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), but not for diffuse large B-cell lymphoma (DLBCL). The molecular basis behind these discrepancies remained unknown. Our aim was to define intracellular signaling events in the induction of direct B-cell death upon rituximab and obinutuzumab treatment. We performed LC-MS/MS phosphoproteomics on SU-DHL4 lymphoma cells treated with rituximab or obinutuzumab (0, 1 and 24 h time-points). Kinase activities were inferred by kinase-substrate enrichment analysis linking kinases to their phosphosite substrates. Immunoblotting was done where necessary to understand the nature of specific signaling events. The activity of 41 and 40 protein kinases was altered upon rituximab or obinutuzumab treatment, respectively, 32 of which were affected by both mAbs. Pathway enrichment analyses revealed up-regulated B-cell receptor (BCR) signaling and down-regulated cell cycle progression with both treatments. To delineate differences between the two mAbs we investigated signaling events in the BCR cascade in more detail. The proximal BCR kinase SYK was strongly phosphorylated on Tyr352 by both mAbs, whereas SYK Tyr525/526 phosphorylation, essential for normal kinase function, was detectable at a low level only with rituximab. Phospho-SYK Tyr352 in the absence of phospho-SYK Tyr525/526 has been associated with autoimmune checkpoint activation and B-cell apoptosis. Both mAbs phosphorylated the key BCR kinase BTK on Tyr551 but not on Tyr223. The latter site was essential for full kinase activity and, in line with incomplete BTK activation, downstream PCLγ2 phosphorylation was delayed. No evidence was found for PLCγ2 dependent de-phosphorylation of NFAT2/NFAT3 and PKCβ activation. Instead, we observed NFAT1 de-phosphorylation and PKCδ activation, both of which have been associated with an anergic B-cell reaction. The MAPK pathway and MYK were strongly activated by both mAbs serving as potent inducers of cell death in the absence of pro-survival signals. To determine if pro-survival signals were generated, we assessed PI3K and NF-κB activation. While we found no evidence for NF-κB activation, differences in the phosphorylation status of PI3K binding sites on CD19 and BCAP suggest stronger PI3K activation by rituximab than obinutuzumab. In keeping with this, the PI3K effector AKT was much more strongly activated by rituximab. AKT activity was likely also increased by Ca2+-flux following rituximab but not obinutuzumab treatment and modified by differential SHIP1 phosphorylation, a phosphatase that hydrolyzes PI3K-generated PIP3. A central AKT target in apoptosis regulation is BAD. We found increased BAD Ser99 and Ser118 phosphorylation only after rituximab treatment. Rituximab thereby sequestered BAD in the cytosol and impaired its inhibitory effects on anti-apoptotic BCL-2 and BCL-xL. Moreover, signaling events protecting against caspase 2 activation were exclusively found for rituximab. In summary, here we show that rituximab and obinutuzumab both induce BCR signaling capable of promoting B-cell apoptosis. However, rituximab generates signals that increase anti-apoptotic BCL-2 effects and diminish B-cell death, whereas obinutuzumab more readily overcomes resistance through BCL-2 overexpression characteristic for FL and CLL. The differences found between rituximab and obinutuzumab are likely less pronounced in cases with strong tonic BCR signaling, which is more prevalent in DLBCL. Thus, our findings provide a mechanistic rationale for the observations made in clinical trials comparing rituximab and obinutuzumab head-to-head. We believe that our results will help to identify new patient subgroups benefitting from obinutuzumab and to better assess the role of anti-CD20 treatment in combination therapies. Disclosures Vilventhraraja: Janssen Pharmaceutical Companies: Employment. Döhner:AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding; Celgene, Novartis, Sunesis: Honoraria, Research Funding. Gribben:Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Fas signaling-mediated TH9 cell differentiation favors bowel inflammation and antitumor functions

Fas induces apoptosis in activated T cell to maintain immune homeostasis, but the effects of non-apoptotic Fas signaling on T cells remain unclear. Here we show that Fas promotes TH9 cell differentiation by activating NF-κB via Ca2+-dependent PKC-β activation. In addition, PKC-β also phosphorylates p38 to inactivate NFAT1 and reduce NFAT1-NF-κB synergy to promote the Fas-induced TH9 transcription program. Fas ligation exacerbates inflammatory bowel disease by increasing TH9 cell differentiation, and promotes antitumor activity in p38 inhibitor-treated TH9 cells. Furthermore, low-dose p38 inhibitor suppresses tumor growth without inducing systemic adverse effects. In patients with tumor, relatively high TH9 cell numbers are associated with good prognosis. Our study thus implicates Fas in CD4+ T cells as a target for inflammatory bowel disease therapy. Furthermore, simultaneous Fas ligation and low-dose p38 inhibition may be an effective approach for TH9 cell induction and cancer therapy.

Remifentanil attenuates lipopolysaccharide-induced oxidative injury by downregulating PKCβ2 activation and inhibiting autophagy in H9C2 cardiomyocytes

AimLipopolysaccharide (LPS)-induced myocardial injury is a leading cause of death in patients with sepsis, which is associated with excessive activation of PKCβ (especially PKCβ2) and autophagy. Remifentanil, a μ-opioid receptor agonist, is well demonstrated to have beneficial effects during sepsis, but the underlying mechanisms are still unknown. The present study was designed to investigate the roles of remifentanil in PKCβ2 and autophagy in LPS-treated cardiomyocytes. Main methodsH9C2 cardiomyocytes were treated with or without remifentanil (2.5 μM), PKCβ2 inhibitor CGP53353 (CGP, 1 μM) or autophagy inhibitor 3-methyladenine (3-MA, 10 μM) in the presence or absence of LPS (10 μg/mL). Key findingsLPS exposure for 24 h led to a significant increase in cell death, LDH release and MDA production in H9C2 cardiomyocytes, accompanied with decreased SOD activity and excessive PKCβ2 activation and autophagy indicated by enhanced Beclin-1 and LC-3II expression and decreased p62 expression. All these changes were attenuated by remifentanil intervention. In addition, inhibition of LPS-induced PKCβ2 activation by CGP or autophagy inhibitor 3-MA has similar effects to remifentanil. SignificanceRemifentanil protects H9C2 cardiomyocytes against LPS-induced oxidative injury, as a result of downregulating PKCβ2 activation and inhibiting autophagy, partially.

Glycine Protects H9C2 Cardiomyocytes from High Glucose- and Hypoxia/Reoxygenation-Induced Injury via Inhibiting PKCβ2 Activation and Improving Mitochondrial Quality.

Background Patients with diabetes are more vulnerable to myocardial ischemia reperfusion injury (IRI), which is involved in PKCβ2 activation and mitochondrial dysfunction. Glycine has been documented as a cytoprotective agent to attenuate diabetes-related abnormalities and reduce myocardial IRI, but the underlying mechanisms are still unclear. We determined whether glycine could attenuate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by inhibiting PKCβ2 activation and improving mitochondrial quality in cultured H9C2 cells. Methods H9C2 cells were either exposed to low glucose (LG) or HG conditions with or without treatment of glycine or CGP53353 (a selective inhibitor of PKCβ2) for 48 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using corresponding commercial kits. Mitochondrial quality control-related proteins (LC-3II, Mfn-2, and Cyt-C) and PKCβ2 activation were detected by Western blot. Results HG stimulation significantly decreased cell viability and SOD activity and increased LDH release, MDA production, and PKCβ2 activation as compared to LG group, all of which changes were further increased by H/R insult. Glycine or CGP53353 treatment significantly reduced the increase of LDH release, MDA production, PKCβ2 activation, and Cyt-C expression and the decrease of cell viability, SOD activity, MMP, Mfn-2 expression, and LC-3II/LC-3I ratio induced by HG and H/R stimulation. Conclusions Supplementary glycine protects H9C2 cells from HG- and H/R-induced cellular injury by suppressing PKCβ2 activation and improving mitochondria quality.

Protein kinase Cβ activates fat mass and obesity-associated protein by influencing its ubiquitin/proteasome degradation.

Protein kinase Cβ (PKCβ) is a serine-threonine kinase associated with obesity and diabetic complications; its activation contributes to weight gain, and deletion of its gene results in resistance to genetic- and diet-induced obesity. Fat mass and obesity-associated (FTO) protein is a recently identified RNA demethylase, and its overexpression in mice leads to increased body weight as well as fat mass. Although sharing some features in anabolism regulation, PKCβ and FTO have not been investigated together; therefore, their relationship has not been established. We report that PKCβ positively regulates FTO on the posttranslation level, evidenced by the facts that PKCβ activation contributes to high-glucose-induced FTO up-regulation, and overexpression of PKCβ suppresses ubiquitin-proteasome degradation of FTO, whereas PKCβ inactivation acts in the opposite manner. It was also found that PKCβ can phosphorylate FTO on threonine, and this phosphorylation requires both catalytic and regulatory domains of PKCβ. Moreover, PKCβ inhibition can suppress 3T3-L1 cell differentiation in normal and FTO-overexpressing cells but not in FTO-silenced or -inhibited cells. We propose that PKCβ acts to suppress the degradation of FTO protein and reveals the associated role of PKCβ and FTO in adipogenesis, suggesting a new pathway that affects the development of obesity and metabolic diseases.-Tai, H., Wang, X., Zhou, J., Han, X., Fang, T., Gong, H., Huang, N., Chen, H., Qin, J., Yang, M., Wei, X., Yang, L., Xiao, H. Protein kinase Cβ activates fat mass and obesity-associated protein by influencing its ubiquitin/proteasome degradation.

DCP-LA, a New Strategy for Alzheimer's Disease Therapy

Alzheimer’s disease (AD) is characterized by extensive deposition of amyloid β (Aβ) and formation of neurofibrillary tangles (NFTs) consisting of hyperphosphorylated Tau. So far, a variety of AD drugs targeting Aβ have been developed, but ended in failure. A recent focus on AD therapy, therefore, is development of Tau-targeted drugs. Aβ activates glycogen synthase kinase-3β (GSK-3β), that plays a central role in Tau phosphorylation, responsible for NFT formation. The linoleic acid derivative DCP-LA has been developed as a promising drug for AD therapy. DCP-LA serves as a selective activator of PKCe and a potent inhibitor of protein tyrosine phosphatase 1B (PTP1B). DCP-LA restrains Tau phosphorylation efficiently due to PKCe-mediated direct inactivation of GSK-3β, to PKCe/Akt-mediated inactivation of GSK-3β, and to receptor tyrosine kinase/insulin receptor substrate 1/phosphoinositide 3-kinase/3-phosphoinositide-dependent protein kinase 1/Akt-mediated inactivation of GSK-3β in association with PTP1B inhibition. Moreover, DCP-LA ameliorates spatial learning and memory impairment in 5xFAD transgenic mice, an animal model of AD. Consequently, combination of PKCβ activation and PTP1B inhibition must be an innovative strategy for AD therapy.