Protein kinase C-delta inhibition is organ-protective, enhances pathogen clearance, and improves survival in sepsis.

Sepsis is a leading cause of morbidity and mortality in intensive care units. Previously, we identified Protein Kinase C-delta (PKCδ) as an important regulator of the inflammatory response in sepsis. An important issue in development of anti-inflammatory therapeutics is the risk of immunosuppression and inability to effectively clear pathogens. In this study, we investigated whether PKCδ inhibition prevented organ dysfunction and improved survival without compromising pathogen clearance. Sprague Dawley rats underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. Post-surgery, PBS or a PKCδ inhibitor (200µg/kg) was administered intra-tracheally (IT). At 24 hours post-CLP, there was evidence of lung and kidney dysfunction. PKCδ inhibition decreased leukocyte influx in these organs, decreased endothelial permeability, improved gas exchange, and reduced blood urea nitrogen/creatinine ratios indicating organ protection. PKCδ inhibition significantly decreased bacterial levels in the peritoneal cavity, spleen and blood but did not exhibit direct bactericidal properties. Peritoneal chemokine levels, neutrophil numbers, or macrophage phenotypes were not altered by PKCδ inhibition. Peritoneal macrophages isolated from PKCδ inhibitor-treated septic rats demonstrated increased bacterial phagocytosis. Importantly, PKCδ inhibition increased survival. Thus, PKCδ inhibition improved survival and improved survival was associated with increased phagocytic activity, enhanced pathogen clearance, and decreased organ injury.

A novel delivery nanobiotechnology: engineered miR-181b exosomes improved osteointegration by regulating macrophage polarization.

BACKGROUND: Many patients suffer from implant loosening after the implantation of titanium alloy caused by immune response to the foreign bodies and this could inhibit the following osteogenesis, which could possibly give rise to aseptic loosening and poor osteointegration while there is currently no appropriate solution in clinical practice. Exosome (Exo) carrying miRNA has been proven to be a suitable nanocarrier for solving this problem. In this study, we explored whether exosomes overexpressing miR-181b (Exo-181b) could exert beneficial effect on promoting M2 macrophage polarization, thus inhibiting inflammation as well as promoting osteogenesis and elaborated the underlying mechanism in vitro. Furthermore, we aimed to find whether Exo-181b could enhance osteointegration. RESULTS: In vitro, we firstly verified that Exo-181b significantly enhanced M2 polarization and inhibited inflammation by suppressing PRKCD and activating p-AKT. Then, in vivo, we verified that Exo-181b enhanced M2 polarization, reduced the inflammatory response and enhanced osteointegration. Also, we verified that the enhanced M2 polarization could indirectly promote the migration and osteogenic differentiation by secreting VEGF and BMP-2 in vitro. CONCLUSIONS: Exo-181b could suppress inflammatory response by promoting M2 polarization via activating PRKCD/AKT signaling pathway, which further promoting osteogenesis in vitro and promote osteointegration in vivo.

Protein Kinase C-δ Mediates Kidney Tubular Injury in Cold Storage-Associated Kidney Transplantation.

BACKGROUND: Kidney injury associated with cold storage is a determinant of delayed graft function and the long-term outcome of transplanted kidneys, but the underlying mechanism remains elusive. We previously reported a role of protein kinase C-δ (PKCδ) in renal tubular injury during cisplatin nephrotoxicity and albumin-associated kidney injury, but whether PKCδ is involved in ischemic or transplantation-associated kidney injury is unknown. METHODS: To investigate PKCδ's potential role in injury during cold storage-associated transplantation, we incubated rat kidney proximal tubule cells in University of Wisconsin (UW) solution at 4°C for cold storage, returning them to normal culture medium at 37°C for rewarming. We also stored kidneys from donor mice in cold UW solution for various durations, followed by transplantation into syngeneic recipient mice. RESULTS: We observed PKCδ activation in both in vitro and in vivo models of cold-storage rewarming or transplantation. In the mouse model, PKCδ was activated and accumulated in mitochondria, where it mediated phosphorylation of a mitochondrial fission protein, dynamin-related protein 1 (Drp1), at serine 616. Drp1 activation resulted in mitochondrial fission or fragmentation, accompanied by mitochondrial damage and tubular cell death. Deficiency of PKCδ in donor kidney ameliorated Drp1 phosphorylation, mitochondrial damage, tubular cell death, and kidney injury during cold storage-associated transplantation. PKCδ deficiency also improved the repair and function of the renal graft as a life-supporting kidney. An inhibitor of PKCδ, δV1-1, protected kidneys against cold storage-associated transplantation injury. CONCLUSIONS: These results indicate that PKCδ is a key mediator of mitochondrial damage and renal tubular injury in cold storage-associated transplantation and may be an effective therapeutic target for improving renal transplant outcomes.

Moracin D induces apoptosis in prostate cancer cells via activation of PPAR gamma/PKC delta and inhibition of PKC alpha.

Herein, apoptotic mechanism of Moracin D was explored in prostate cancer cells in association with peroxisome proliferator-activated receptor gamma (PPAR-γ)-related signaling involved in lipid metabolism. Moracin D augmented cytotoxicity and sub G1 population in PC3 and DU145 prostate cancer cells, while DU145 cells were more susceptible to Moracin D than PC3 cells. Moracin D attenuated the expression of caspase-3, poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), and B-cell lymphoma-extra-large (Bcl-xL) in DU145 cells. Consistently, Moracin D significantly augmented the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in DU145 cells. Interestingly, Moracin D activated PPAR-γ and phospho-protein kinase C delta (p-PKC-δ) and inhibited phospho-protein kinase C alpha (p-PKC-α) in DU145 cells. Furthermore, STRING bioinformatic analysis reveals that PPAR-γ interacts with nuclear factor-κB (NF-κB) that binds to PKC-α/PKC-δ or protein kinase B (AKT) or extracellular signal-regulated kinase (ERK). Indeed, Moracin D decreased phosphorylation of NF-κB, ERK, and AKT in DU145 cells. Conversely, PPAR-γ inhibitor GW9662 reduced the apoptotic ability of Moracin D to activate caspase 3 and PARP in DU145 cells. Taken together, these findings provide a novel insight that activation of PPAR-γ/p-PKC-δ and inhibition of p-PKC-α are critically involved in Moracin D-induced apoptosis in DU145 prostate cancer cells.

A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals.

The metabolic consequences and sequelae of obesity promote life-threatening morbidities. PKCδI is an important elicitor of inflammation and apoptosis in adipocytes. Here we report increased PKCδI activation via release of its catalytic domain concurrent with increased expression of proinflammatory cytokines in adipocytes from obese individuals. Using a screening strategy of dual recognition of PKCδI isozymes and a caspase-3 binding site on the PKCδI hinge domain with Schrödinger software and molecular dynamics simulations, we identified NP627, an organic small-molecule inhibitor of PKCδI. Characterization of NP627 by surface plasmon resonance (SPR) revealed that PKCδI and NP627 interact with each other with high affinity and specificity, SPR kinetics revealed that NP627 disrupts caspase-3 binding to PKCδI, and in vitro kinase assays demonstrated that NP627 specifically inhibits PKCδI activity. The SPR results also indicated that NP627 affects macromolecular interactions between protein surfaces. Of note, release of the PKCδI catalytic fragment was sufficient to induce apoptosis and inflammation in adipocytes. NP627 treatment of adipocytes from obese individuals significantly inhibited PKCδI catalytic fragment release, decreased inflammation and apoptosis, and significantly improved mitochondrial metabolism. These results indicate that PKCδI is a robust candidate for targeted interventions to manage obesity-associated chronic inflammatory diseases. We propose that NP627 may also be used in other biological systems to better understand the impact of caspase-3-mediated activation of kinase activity.

Longifolioside A inhibits TLR4-mediated inflammatory responses by blocking PKCδ activation in LPS-stimulated THP-1 macrophages.

Longifolioside A is an iridoid glucoside compound isolated from Pseudolysimachion rotundum var. subintegrum, which has been used in traditional herbal medicines to treat respiratory inflammatory diseases. Logifolioside A is a potent antioxidant; however, its underlying pharmacological mechanisms of action in inflammatory diseases are unknown. Here, we investigated the inhibitory effects of longifolioside A in lipopolysaccharide (LPS)-stimulated toll-like receptor 4 (TLR4) signal transduction systems using human THP-1 macrophages and HEK293 cells stably expressing human TLR4 protein (293/HA-hTLR4). Longifolioside A significantly reduced the release of inflammatory cytokines such as interleukin (IL)-6, -8, and tumor necrosis factor (TNF)-α in LPS-stimulated THP-1 macrophages. Furthermore, longifolioside A inhibited the expression of inflammatory mediator genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 that produce nitric oxide (NO) and prostaglandin E2 (PGE2), respectively. Longifolioside A suppressed the phosphorylation of PKCδ, IRAK4, IKKα/ß, IκBα, and mitogen-activated protein (MAP) kinases (ERK 1/2 and JNK, but not p38), thereby inactivating the nuclear localization of NF-κB and AP-1, and thus decreasing the expression of inflammatory response genes. Notably, longifolioside A disrupted the interaction between human TLR4 and the TIR domain-containing adaptor protein (TIRAP), an early step during TLR4 activation, thereby reducing IL-8 secretion in 293/HA-hTLR4 cells. This inhibitory effect was comparable to that of TAK-242 (a TLR4 inhibitor, or resatorvid). Our results indicate that longifolioside A prevents inflammatory response by suppressing TLR4 activation required for NF-κB and AP-1 activation.

Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1ß in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.

Prenatal exposure to testosterone induces cardiac hypertrophy in adult female rats through enhanced Pkcδ expression in cardiac myocytes.

High circulating androgen in women with polycystic ovary syndrome (PCOS) may increase the risk of cardiovascular disease in offspring. The aim of the present study is to investigate whether maternal androgen excess in the rat PCOS model would lead to cardiac hypertrophy in offspring. Maternal testosterone propionate (maternal-TP)-treated adult female offspring displayed cardiac hypertrophy associated with local high cardiac dihydrotestosterone (DHT). The molecular markers of cardiac hypertrophy along with androgen receptor (AR) and PKCδ, were increased in the Maternal-TP group. Treatment of primary neonatal rat ventricular cardiomyocytes (NRCMs) and H9c2 cells with DHT significantly increased cell size and upregulated PKCδ expression, which could be attenuated by AR antagonist. Treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly increased cell size and upregulated myh7 level. Rottlerin, that may inhibit PKCδ, significantly reduced the hypertrophic effect of DHT and PMA on NRCMs and H9c2 cells. Chromatin immunoprecipitation revealed that AR could bind to Pkcδ promoter. Our results indicate that prenatal exposure to testosterone may induce cardiac hypertrophy in adult female rats through enhanced Pkcδ expression in cardiac myocytes.

Protein kinase Cδ constrains the S-pathway to phrenic motor facilitation elicited by spinal 5-HT7 receptors or severe acute intermittent hypoxia.

KEY POINTS: Concurrent 5-HT2A (Q pathway) and 5-HT7 (S pathway) serotonin receptor activation cancels phrenic motor facilitation due to mutual cross-talk inhibition. Spinal protein kinase Cδ (PKCδ) or protein kinase A inhibition restores phrenic motor facilitation with concurrent Q and S pathway activation, demonstrating a key role for these kinases in cross-talk inhibition. Spinal PKCδ inhibition enhances adenosine-dependent severe acute intermittent hypoxia-induced phrenic long-term facilitation (S pathway), consistent with relief of cross-talk inhibition. ABSTRACT: Intermittent spinal serotonin receptor activation elicits long-lasting phrenic motor facilitation (pMF), a form of respiratory motor plasticity. When activated alone, spinal Gq protein-coupled serotonin 2A receptors (5-HT2A ) initiate pMF by a mechanism that requires ERK-MAP kinase signalling and new BDNF protein synthesis (Q pathway). Spinal Gs protein-coupled serotonin 7 (5-HT7 ) and adenosine 2A (A2A ) receptor activation also elicits pMF, but via distinct mechanisms (S pathway) that require Akt signalling and new TrkB protein synthesis. Although studies have shown inhibitory cross-talk interactions between these competing pathways, the underlying cellular mechanisms are unknown. We propose the following hypotheses: (1) concurrent 5-HT2A and 5-HT7 activation undermines pMF; (2) protein kinase A (PKA) and (3) NADPH oxidase mediate inhibitory interactions between Q (5-HT2A ) and S (5-HT7 ) pathways. Selective 5-HT2A (DOI hydrochloride) and 5HT7 (AS-19) agonists were administered intrathecally at C4 (three injections, 5-min intervals) in anaesthetized, vagotomized and ventilated male rats. With either spinal 5-HT2A or 5-HT7 activation alone, phrenic amplitude progressively increased (pMF). In contrast, concurrent 5-HT2A and 5-HT7 activation failed to elicit pMF. The 5-HT2A -induced Q pathway was restored by inhibiting PKA activity (Rp-8-Br-cAMPS). NADPH oxidase inhibition did not prevent cross-talk inhibition. Therefore, we investigated alternative mechanisms to explain Q to S pathway inhibition. Spinal protein kinase C (PKC) inhibition with Gö6983 or PKCδ peptide inhibitor restored the 5-HT7 -induced S pathway to pMF, revealing PKCδ as the relevant isoform. Spinal PKCδ inhibition enhanced the S pathway-dependent form of pMF elicited by severe acute intermittent hypoxia. We suggest that powerful constraints between 5-HT2A and 5-HT7 or A2A receptor-induced pMF are mediated by PKCδ and PKA, respectively.

Protein kinase C-delta inhibition protects blood-brain barrier from sepsis-induced vascular damage.

BACKGROUND: Neuroinflammation often develops in sepsis leading to activation of cerebral endothelium, increased permeability of the blood-brain barrier (BBB), and neutrophil infiltration. We have identified protein kinase C-delta (PKCδ) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKCδ by a peptide inhibitor (PKCδ-i) protected endothelial cells, decreased sepsis-mediated neutrophil influx into the lung, and prevented tissue damage. The objective of this study was to elucidate the regulation and relative contribution of PKCδ in the control of individual steps in neuroinflammation during sepsis. METHODS: The role of PKCδ in mediating human brain microvascular endothelial (HBMVEC) permeability, junctional protein expression, and leukocyte adhesion and migration was investigated in vitro using our novel BBB on-a-chip (B3C) microfluidic assay and in vivo in a rat model of sepsis induced by cecal ligation and puncture (CLP). HBMVEC were cultured under flow in the vascular channels of B3C. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF-α (10 U/ml) for 4 h in the absence or presence of PKCδ-i (5 µM) to quantify neutrophil adhesion and migration in the B3C. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo. RESULTS: During sepsis, PKCδ is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKCδ-i. Similarly, TNF-α-mediated activation of PKCδ and its translocation in HBMVEC are attenuated by PKCδ-i in vitro. PKCδ inhibition significantly reduced TNF-α-mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24 h after CLP induced sepsis. TNF-α-treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKCδ-i-treated cells. PKCδ inhibition also reduced TNF-α-mediated neutrophil adhesion and migration across HBMVEC in B3C. Interestingly, while PKCδ inhibition decreased the number of adherent neutrophils to baseline (no-treatment group), it significantly reduced the number of migrated neutrophils below the baseline, suggesting a critical role of PKCδ in regulating neutrophil transmigration. CONCLUSIONS: The BBB on-a-chip (B3C) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKCδ activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKCδ-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage.

Exposure to far-infrared ray attenuates methamphetamine-induced impairment in recognition memory through inhibition of protein kinase C δ in male mice: Comparison with the antipsychotic clozapine.

We have previously demonstrated that repeated treatment with methamphetamine (MA) results in a recognition memory impairment via upregulation of protein kinase C (PKC) δ and downregulation of the glutathione peroxidase-1 (GPx-1)-dependent antioxidant system. We also demonstrated that far-infrared ray (FIR) attenuates acute restraint stress via induction of the GPx-1 gene. Herein, we investigated whether exposure to FIR modulates MA-induced recognition memory impairment in male mice, and whether cognitive potentials mediated by FIR require modulation of the PKCδ gene, extracellular signal-regulated kinase (ERK) 1/2, and glutathione-dependent system. Repeated treatment with MA significantly increased PKCδ expression and its phosphorylation out of PKC isoenzymes (i.e., PKCα, PKCßI, PKCßII, PKCζ, and PKCδ expression) in the prefrontal cortex of mice. Exposure to FIR significantly attenuated MA-induced increase in phospho-PKCδ and decrease in phospho-ERK 1/2. In addition, FIR further facilitated the nuclear factor E2-related factor 2 (Nrf2)-dependent glutathione synthetic system. Moreover, L-buthionine-(S, R)-sulfoximine, an inhibitor of glutathione synthesis, counteracted the FIR-mediated phospho-ERK 1/2 induction and memory-enhancing activity against MA insult. More important, positive effects of FIR are comparable to those of genetic depletion of PKCδ or the antipsychotic clozapine. Our results indicate that FIR protects against MA-induced memory impairment via activations of the Nrf2-dependent glutathione synthetic system, and ERK 1/2 signaling by inhibition of the PKCδ gene.

Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase.

OBJECTIVE: Impaired endothelial cell (EC) autophagy compromises shear stress-induced nitric oxide (NO) generation. We determined the responsible mechanism. APPROACH AND RESULTS: On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOSS1177) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOSS1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδT505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress-induced activating phosphorylation of PKCδT505 is negated by inhibiting autophagy, (2) shear-induced p-eNOSS1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKCδ) activation of PKCδT505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKCδ small interfering RNA) PKCδ inhibition prevents shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs. CONCLUSIONS: Targeted reactivation of purinergic signaling and PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

Protein kinase Cδ (PKCδ) involved in the regulation of pAkt1 (Ser473) on the release of mouse oocytes from diplotene arrest.

Defects in meiotic maturation may lead to chromosome segregation errors and genomic instability. Previous reports indicated that protein kinase C delta (PKCδ) may interact with microtubule organizing center-associated protein to play a role on meiotic spindle organization of mammalian oocytes. In this study, we explored the potential role of protein kinase B alpha (PKBα/Akt1), PKCδ, and their relationship on meiotic maturation of mouse oocytes. We examined the expression and localization of pAkt1 (Ser473) and/or pPKCδ (Thr505) under the treatment of SH-6 or Sotrastaurin. With the increasing of SH-6 concentrations, we observed that the protein levels of pAkt1 (Ser473) and the percentages of GVBD were decreased gradually. And the distribution of pAkt1 (Ser473), Cdc25B, pCdc2 (Tyr15), and α-tubulin around nucleus was also highly disordered under the treatment of 10-µM SH-6, a special inhibitor of pAkt1 (Ser473). In addition, the levels of pAkt1 (Ser473) were decreased with the treatment of Sotrastaurin, an inhibitor of PKCδ, suggesting that Akt1 may be one of the downstream targets of PKCδ. So, we deduced that PKCδ may be involved in regulating the release from diplotene arrest of mouse oocytes by controlling the levels of pAkt1 Ser473. SIGNIFICANCE OF THE STUDY: As a drug target, Akt is still the focus of research in somatic and fertilized eggs. Here, our data depicted a series of molecular events of pAkt1 (Ser473) and PKCδ via their special inhibitor and antibody in order to analyse their role and relationship on the release of mouse oocytes from diplotene arrest. Our results may offer important data for clinic research scientist to recognize the target role of pAkt1 (Ser473) via PKCδ in cell cycle regulation.

Pro-Inflammatory Stimuli Influence Expression of Intercellular Adhesion Molecule 1 in Human Anulus Fibrosus Cells through FAK/ERK/GSK3 and PKCδ Signaling Pathways.

OBJECTIVE: Intervertebral disc (IVD) degeneration and disc herniation are major causes of lower back pain, which involve the presence of inflammatory mediators and tissue invasion by immune cells. Intercellular adhesion molecule 1 (ICAM1, also termed CD54) is an adhesion molecule that mediates cell-cell interactions, particularly between immune cells and target tissue. The aim of this study was to examine the intracellular signaling pathways involved in inflammatory stimuli-induced ICAM1 expression in human anulus fibrosus (AF) cells. METHODS: Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and flow cytometry were performed to dissect the roles of different signaling pathways in inflammatory stimuli-mediated ICAM1 expression. RESULTS: Using qPCR and western blot analyses, a significant increase in ICAM1 expression was observed in AF cells after stimulation of lipopolysaccharide (LPS) plus interferon-gamma (IFNγ) in a time-dependent manner. Flow cytometry revealed ICAM1 upregulation on the surface of AF cells. Importantly, LPS plus IFNγ treatment also significantly promoted Chemokine ligand (CCL)2 expression, but not CCL3. The enhanced ICAM1 expression was abolished after incubation with antibody against CCL2. In AF cells, treatment with LPS plus IFNγ activated the FAK/ERK/GSK3 signaling pathways, promoted a time-dependent increase in PKCδ phosphorylation, and promoted PKCδ translocation to the nucleus. Treatment with the pharmacological PKCδ inhibitor; rottlerin, effectively blocked the enhanced productions of ICAM1 and CCL2. CONCLUSIONS: Inflammatory stimuli in AF cells are part of a specific pathophysiology in IVD degeneration and disc herniation that modulates CCL2/ICAM1 activation through the FAK/ERK/GSK3 and PKCδ signaling pathways in AF cells.

Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ.

Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1ß, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.

Long chain n-3 polyunsaturated fatty acids increase the efficacy of docetaxel in mammary cancer cells by downregulating Akt and PKCε/δ-induced ERK pathways.

Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.

Spinal BDNF-induced phrenic motor facilitation requires PKCθ activity.

Spinal brain-derived neurotrophic factor (BDNF) is necessary and sufficient for certain forms of long-lasting phrenic motor facilitation (pMF). BDNF elicits pMF by binding to its high-affinity receptor, tropomyosin receptor kinase B (TrkB), on phrenic motor neurons, potentially activating multiple downstream signaling cascades. Canonical BDNF/TrkB signaling includes the 1) Ras/RAF/MEK/ERK MAP kinase, 2) phosphatidylinositol 3-kinase (PI3K)/Akt, and 3) PLCγ/PKC pathways. Here we demonstrate that spinal BDNF-induced pMF requires PLCγ/PKCθ in normal rats but not MEK/ERK or PI3K/Akt signaling. Cervical intrathecal injections of MEK/ERK (U0126) or PI3K/Akt (PI-828; 100 µM, 12 µl) inhibitor had no effect on BDNF-induced pMF (90 min after BDNF; U0126 + BDNF: 59 ± 14%, PI-828 + BDNF: 59 ± 8%, inhibitor vehicle + BDNF: 56 ± 7%; all P ≥ 0.05). In contrast, PKCθ inhibition with theta inhibitory peptide (TIP; 0.86 mM, 12 µl) prevented BDNF-induced pMF (90 min after BDNF; TIP + BDNF: -2 ± 2%; P ≤ 0.05 vs. other groups). Thus BDNF-induced pMF requires downstream PLCγ/PKCθ signaling, contrary to initial expectations.NEW AND NOTEWORTHY We demonstrate that BDNF-induced pMF requires downstream signaling via PKCθ but not MEK/ERK or PI3K/Akt signaling. These data are essential to understand the sequence of the cellular cascade leading to BDNF-dependent phrenic motor plasticity.

A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog.

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells. Ablation of Hdac1 or Hdac2 by Nestin-Cre had no obvious consequences on brain development and architecture owing to compensation by the paralog. By contrast, combined deletion of Hdac1 and Hdac2 resulted in impaired chromatin structure, DNA damage, apoptosis and embryonic lethality. To dissect the individual roles of HDAC1 and HDAC2, we expressed single alleles of either Hdac1 or Hdac2 in the absence of the respective paralog in neural cells. The DNA-damage phenotype observed in double knockout brains was prevented by expression of a single allele of either Hdac1 or Hdac2. Strikingly, Hdac1(-/-)Hdac2(+/-) brains showed normal development and no obvious phenotype, whereas Hdac1(+/-)Hdac2(-/-) mice displayed impaired brain development and perinatal lethality. Hdac1(+/-)Hdac2(-/-) neural precursor cells showed reduced proliferation and premature differentiation mediated by overexpression of protein kinase C, delta, which is a direct target of HDAC2. Importantly, chemical inhibition or knockdown of protein kinase C delta was sufficient to rescue the phenotype of neural progenitor cells in vitro. Our data indicate that HDAC1 and HDAC2 have a common function in maintaining proper chromatin structures and show that HDAC2 has a unique role by controlling the fate of neural progenitors during normal brain development.

Regulation of ribosomal RNA synthesis in T cells: requirement for GTP and Ebp1.

Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, an effective immunosuppressive drug. Both MPA and mycophenolate mofetil are highly specific inhibitors of guanine nucleotide synthesis and of T-cell activation. However, the mechanism by which guanine nucleotide depletion suppresses T-cell activation is unknown. Depletion of GTP inhibits ribosomal RNA synthesis in T cells by inhibiting transcription initiation factor I (TIF-IA), a GTP-binding protein that recruits RNA polymerase I to the ribosomal DNA promoter. TIF-IA-GTP binds the ErbB3-binding protein 1, and together they enhance the transcription of proliferating cell nuclear antigen (PCNA). GTP binding by TIF-IA and ErbB3-binding protein 1 phosphorylation by protein kinase C δ are both required for optimal PCNA expression. The protein kinase C inhibitor sotrastaurin markedly potentiates the inhibition of ribosomal RNA synthesis, PCNA expression, and T-cell activation induced by MPA, suggesting that the combination of the two agents are more highly effective than either alone in inducing immunosuppression.

Angiotensin II-preconditioning is associated with increased PKCε/PKCδ ratio and prosurvival kinases in mitochondria.

Angiotensin II-preconditioning (APC) has been shown to reproduce the cardioprotective effects of ischaemic preconditioning (IPC), however, the molecular mechanisms mediating the effects of APC remain unknown. In this study, Langendorff-perfused rat hearts were subjected to IPC, APC or both (IPC/APC) followed by ischaemia-reperfusion (IR), to determine translocation of PKCε, PKCδ, Akt, Erk1/2, JNK, p38 MAPK and GSK-3ß to mitochondria as an indicator of activation of the protein kinases. In agreement with previous observations, IPC, APC and IPC/APC increased the recovery of left ventricular developed pressure (LVDP), reduced infarct size (IS) and lactate dehydrogenase (LDH) release, compared to controls. These effects were associated with increased mitochondrial PKCε/PKCδ ratio, Akt, Erk1/2, JNK, and inhibition of permeability transition pore (mPTP) opening. Chelerythrine, a pan-PKC inhibitor, abolished the enhancements of PKCε but increased PKCδ expression, and inhibited Akt, Erk1/2, and JNK protein levels. The drug had no effect on the APC- and IPC/APC-induced cardioprotection as previously reported, but enhanced the post-ischaemic LVDP in controls. Losartan, an angiotensin II type 1 receptor (AT1-R) blocker, abolished the APC-stimulated increase of LVDP and reduced PKCε, Akt, Erk1/2, JNK, and p38. Both drugs reduced ischaemic contracture and LDH release, and abolished the inhibition of mPTP by the preconditioning. Chelerythrine also prevented the reduction of IS by APC and IPC/APC. These results suggest that the cardioprotection induced by APC and IPC/APC involves an AT1-R-dependent translocation of PKCε and survival kinases to the mitochondria leading to mPTP inhibition. In chelerythrine-treated hearts, however, alternate mechanisms appear to maintain cardiac function.