Structure, biosynthesis and activity of indolactam alkaloids.

Indolactam alkaloids are a family of aromatic toxins produced by various actinobacteria and the cyanobacterium, Moorena producens. The best characterized examples include the teleocidins, lyngbyatoxins, olivoretins, blastmycetins, and pendolmycins, which share a nine-membered lactam core, comprised from l-tryptophanol and l-valine. Contact with indolactam alkaloids has been linked to severe dermatitis (swimmers itch), while accidental ingestion may lead to illness and fatalities. Indolactam alkaloids are also potent tumor promotors, due to their activation of protein kinase C isozymes. This chapter reviews the current literature on indolactam alkaloids, from their discovery in the early 1960s up to 2024. Topics covered include the isolation, structural elucidation, biosynthesis, bioactivity, and total synthesis of the indolactam alkaloid core.

Protein kinase C (PKC) inhibitor calphostin C activates PKC in a light-dependent manner at high concentrations via the production of singlet oxygen.

Calphostin C (Cal-C) is a protein kinase C (PKC) inhibitor that binds to its C1 domain. The aim of the present study was to elucidate the action of Cal-C in addition to PKC inhibition. First, we confirmed that Cal-C at low concentrations (< 200 nM) inhibit phorbol ester-induced PKC translocation and G-protein-coupled receptor (GPCR)-mediated PKC activation. Cal-C at higher concentrations (> 2 µM) increased intracellular calcium ion concentrations ([Ca2+]i) in a concentration-dependent manner. The origin of this increase is the mobilization of the endoplasmic reticulum (ER), which does not involve GPCR or ryanodine receptors. Cal-C at high concentrations also cause structural changes in the ER, such as the formation of vacuoles and aggregates, and calcium leakage from the ER. At 2 µM, Cal-C translocated a calcium-sensitive PKCα. Studies using a C-kinase activity reporter and a myristoylated alanine-rich protein kinase C substrate fused with green fluorescent protein (GFP) have also revealed that Cal-C at high concentrations activate PKC in living cells. Additionally, the PKC-activating effects of Cal-C were light-dependent. Finally, studies using Si-DMA, an indicator of singlet oxygen, showed that Cal-C at high concentrations generated singlet oxygen, causing structural changes in the ER and leakage of calcium into the cytosol, which triggered PKC activation. This study confirms the novel action of Cal-C, solely considered a PKC inhibitor. Cal-C acted as a PKC inhibitor at low concentrations and a PKC activator at high concentrations by generating singlet oxygen in a light-dependent manner, suggesting that Cal-C can be used in photodynamic therapy.

Reduced protein kinase C delta in a high molecular weight complex in mitochondria and elevated creatine uptake into Barth syndrome B lymphoblasts.

Aim: Barth syndrome (BTHS) is a rare X-linked genetic disease in which mitochondrial oxidative phosphorylation is impaired due to a mutation in the TAFAZZIN gene. The protein kinase C delta (PKCδ) signalosome exists as a high molecular weight complex in mitochondria and controls mitochondrial oxidative phosphorylation. Method: Here, we examined PKCδ levels in mitochondria of aged-matched control and BTHS patient B lymphoblasts and its association with a higher molecular weight complex in mitochondria. Result: Immunoblot analysis of blue-native polyacrylamide gel electrophoresis mitochondrial fractions revealed an increase in total PKCδ protein expression in BTHS lymphoblasts compared to controls. In contrast, PKCδ associated with a higher molecular weight complex was markedly reduced in BTHS patient B lymphoblasts compared to controls. Given the decrease in PKCδ associated with a higher molecular weight complex in mitochondria, we examined the uptake of creatine, a compound whose utilization is enhanced upon high energy demand. Creatine uptake was markedly elevated in BTHS lymphoblasts compared to controls. Conclusion: We hypothesize that reduced PKCδ within this higher molecular weight complex in mitochondria may contribute to the bioenergetic defects observed in BTHS lymphoblasts and that enhanced creatine uptake may serve as one of several compensatory mechanisms for the defective mitochondrial oxidative phosphorylation observed in these cells.

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Background: Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/ß-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Methods: Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and ß-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Results: Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and ß-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. Conclusions: PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on ß-catenin in the canonical Wnt pathway was limited. ß-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

A peptide against the N-terminus of myristoylated alanine-rich C kinase substrate promotes neuronal differentiation in SH-SY5Y human neuroblastoma cells.

Myristoylated alanine-rich protein kinase C substrate (MARCKS) plays crucial roles in neuronal functions and differentiation. However, specific effects of the myristoylated N-terminal sequence (MANS) peptide, a widely used MARCKS modulator comprising the initial 24 amino acids of MARCKS, on neuronal cells remain unclear. Therefore, in this study, we aimed to examine the effects and action mechanisms of the MANS peptide on SH-SY5Y human neuroblastoma cells, which served as the in vitro neuronal cell models. MANS treatment of SH-SY5Y cells resulted in significant neurite outgrowth within 24 hr, which was as prominent as that induced by seven days of treatment with all-trans retinoic acid, the most common agent used to induce SH-SY5Y cell differentiation. Levels of synaptophysin, a neuronal marker protein, were significantly increased in the MANS peptide-treated cells. Additionally, increased MARCKS levels and decreased MARCKS phosphorylation were observed in MANS peptide-treated cells. Notably, neurite outgrowth induced by the MANS peptide was significantly reduced in MARCKS-knocked-down cells. Overall, these results suggest the MANS peptide as a novel agent for SH-SY5Y cell differentiation, particularly for the analysis of MARCKS functions.

Ceramide synthesis inhibitors prevent lipid-induced insulin resistance through the DAG-PKCε-insulin receptorT1150 phosphorylation pathway.

Inhibition of the ceramide synthetic pathway with myriocin or an antisense oligonucleotide (ASO) targeting dihydroceramide desaturase (DES1) both improved hepatic insulin sensitivity in rats fed either a saturated or unsaturated fat diet and was associated with reductions in both hepatic ceramide and plasma membrane (PM)-sn-1,2-diacylglycerol (DAG) content. The insulin sensitizing effects of myriocin and Des1 ASO were abrogated by acute treatment with an ASO against DGAT2, which increased hepatic PM-sn-1,2-DAG but not hepatic C16 ceramide content. Increased PM-sn-1,2-DAG content was associated with protein kinase C (PKC)ε activation, increased insulin receptor (INSR)T1150 phosphorylation leading to reduced insulin-stimulated INSRY1152/AktS473 phosphorylation, and impaired insulin-mediated suppression of endogenous glucose production. These results demonstrate that inhibition of de novo ceramide synthesis by either myriocin treatment or DES1 knockdown protects against lipid-induced hepatic insulin resistance through a C16 ceramide-independent mechanism and that they mediate their effects to protect from lipid-induced hepatic insulin resistance via the PM-sn-1,2-DAG-PKCε-INSRT1150 phosphorylation pathway.

Nonalcoholic Fatty Liver Disease and Staging of Hepatic Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is in parallel with the obesity epidemic, and it is the most common cause of liver diseases. The patients with severe insulin-resistant diabetes having high body mass index (BMI), high-grade adipose tissue insulin resistance, and high hepatocellular triacylglycerols (triglycerides; TAG) content develop hepatic fibrosis within a 5-year follow-up. Insulin resistance with the deficiency of insulin receptor substrate-2 (IRS-2)-associated phosphatidylinositol 3-kinase (PI3K) activity causes an increase in intracellular fatty acid-derived metabolites such as diacylglycerol (DAG), fatty acyl CoA, or ceramides. Lipotoxicity-related mechanism of NAFLD could be explained still best by the "double-hit" hypothesis. Insulin resistance is the major mechanism in the development and progression of NAFLD/nonalcoholic steatohepatitis (NASH). Metabolic oxidative stress, autophagy, and inflammation induce NASH progression. In the "first hit" the hepatic concentrations of diacylglycerol increase with an increase in saturated liver fat content in human NAFLD. Activities of mitochondrial respiratory chain complexes are decreased in the liver tissue of patients with NASH. Hepatocyte lipoapoptosis is a critical feature of NASH. In the "second hit," reduced glutathione levels due to oxidative stress lead to the overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling that induces cell death in the steatotic liver. Accumulation of toxic levels of reactive oxygen species (ROS) is caused at least by two ineffectual cyclical pathways. First is the endoplasmic reticulum (ER) oxidoreductin (Ero1)-protein disulfide isomerase oxidation cycle through the downstream of the inner membrane mitochondrial oxidative metabolism and the second is the Kelch like-ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. In clinical practice, on ultrasonographic examination, the elevation of transaminases, γ-glutamyltransferase, and the aspartate transaminase to platelet ratio index indicates NAFLD. Fibrosis-4 index, NAFLD fibrosis score, and cytokeratin18 are used for grading steatosis, staging fibrosis, and discriminating the NASH from simple steatosis, respectively. In addition to ultrasonography, "controlled attenuation parameter," "magnetic resonance imaging proton-density fat fraction," "ultrasound-based elastography," "magnetic resonance elastography," "acoustic radiation force impulse elastography imaging," "two-dimensional shear-wave elastography with supersonic imagine," and "vibration-controlled transient elastography" are recommended as combined tests with serum markers in the clinical evaluation of NAFLD. However, to confirm the diagnosis of NAFLD, a liver biopsy is the gold standard. Insulin resistance-associated hyperinsulinemia directly accelerates fibrogenesis during NAFLD development. Although hepatocyte lipoapoptosis is a key driving force of fibrosis progression, hepatic stellate cells and extracellular matrix cells are major fibrogenic effectors. Thereby, these are pharmacological targets of therapies in developing hepatic fibrosis. Nonpharmacological management of NAFLD mainly consists of two alternatives: lifestyle modification and metabolic surgery. Many pharmacological agents that are thought to be effective in the treatment of NAFLD have been tried, but due to lack of ability to attenuate NAFLD, or adverse effects during the phase trials, the vast majority could not be licensed.

Endothelial Dysfunction in Obesity and Therapeutic Targets.

Parallel to the increasing prevalence of obesity in the world, the mortality from cardiovascular disease has also increased. Low-grade chronic inflammation in obesity disrupts vascular homeostasis, and the dysregulation of adipocyte-derived endocrine and paracrine effects contributes to endothelial dysfunction. Besides the adipose tissue inflammation, decreased nitric oxide (NO)-bioavailability, insulin resistance (IR), and oxidized low-density lipoproteins (oxLDLs) are the main factors contributing to endothelial dysfunction in obesity and the development of cardiorenal metabolic syndrome. While normal healthy perivascular adipose tissue (PVAT) ensures the dilation of blood vessels, obesity-associated PVAT leads to a change in the profile of the released adipo-cytokines, resulting in a decreased vasorelaxing effect. Higher stiffness parameter ß, increased oxidative stress, upregulation of pro-inflammatory cytokines, and nicotinamide adenine dinucleotide phosphate (NADP) oxidase in PVAT turn the macrophages into pro-atherogenic phenotypes by oxLDL-induced adipocyte-derived exosome-macrophage crosstalk and contribute to the endothelial dysfunction. In clinical practice, carotid ultrasound, higher leptin levels correlate with irisin over-secretion by human visceral and subcutaneous adipose tissues, and remnant cholesterol (RC) levels predict atherosclerotic disease in obesity. As a novel therapeutic strategy for cardiovascular protection, liraglutide improves vascular dysfunction by modulating a cyclic adenosine monophosphate (cAMP)-independent protein kinase A (PKA)-AMP-activated protein kinase (AMPK) pathway in PVAT in obese individuals. Because the renin-angiotensin-aldosterone system (RAAS) activity, hyperinsulinemia, and the resultant IR play key roles in the progression of cardiovascular disease in obesity, RAAS-targeted therapies contribute to improving endothelial dysfunction. By contrast, arginase reciprocally inhibits NO formation and promotes oxidative stress. Thus, targeting arginase activity as a key mediator in endothelial dysfunction has therapeutic potential in obesity-related vascular comorbidities. Obesity-related endothelial dysfunction plays a pivotal role in the progression of type 2 diabetes (T2D). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (thiazolidinedione), is a popular drug for treating diabetes; however, it leads to increased cardiovascular risk. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin (EMPA) significantly improves endothelial dysfunction and mortality occurring through redox-dependent mechanisms. Although endothelial dysfunction and oxidative stress are alleviated by either metformin or EMPA, currently used drugs to treat obesity-related diabetes neither possess the same anti-inflammatory potential nor simultaneously target endothelial cell dysfunction and obesity equally. While therapeutic interventions with glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide or bariatric surgery reverse regenerative cell exhaustion, support vascular repair mechanisms, and improve cardiometabolic risk in individuals with T2D and obesity, the GLP-1 analog exendin-4 attenuates endothelial endoplasmic reticulum stress.

Protein kinase C iota promotes glycolysis via PI3K/AKT/mTOR signalling in high grade serous ovarian cancer.

BACKGROUND: Epithelial ovarian cancer, especially high grade serous ovarian cancer (HGSOC) is by far, the most lethal gynecological malignancy with poor prognosis and high relapse rate. Despite of availability of several therapeutic interventions including poly-ADP ribose polymerase (PARP) inhibitors, HGSOC remains unmanageable and identification of early detection biomarkers and therapeutic targets for this lethal malady is highly warranted. Aberrant expression of protein kinase C iota (PKCί) is implicated in many cellular and physiological functions involved in tumorigenesis including cell proliferation and cell cycle deregulation. METHODS AND RESULTS: Two high grade serous ovarian cancer cells SKOV3 and COV362 were employed in this study. PKCί was genetically knocked down or pharmacologically inhibited and several functional and biochemical assays were performed. We report that PKCί is overexpressed in HGSOC cells and patient tissue samples with a significant prognostic value. Pharmacological inhibition of PKCί by Na-aurothiomalate or its shRNA-mediated genetic knockdown suppressed HGSOC cell proliferation, EMT and induced apoptosis. Moreover, PKCί positively regulated GLUT1 and several other glycolytic genes including HK1, HK2, PGK1, ENO1 and LDHA to promote elevated glucose uptake and glycolysis in HGSOC cells. Mechanistically, PKCί drove glycolysis via PI3K/AKT/mTOR signalling. Na-aurothiomalate and highly selective, dual PI3K/mTOR inhibitor dactolisib could serve as novel anti-glycolytic drugs in HGSOC. CONCLUSION: Taken together, our results indicate PKCί/PI3K/AKT/mTOR signalling cascade could be a novel therapeutic target in a lethal pathology like HGSOC.

Neuroprotective effects of ulinastatin on Escherichia coli meningitis rats through inhibiting PKCα phosphorylation and reducing zonula occludens-1 degradation.

Ulinastatin, a broad-spectrum inflammatory inhibitor widely employed in the management of severe pancreatitis and sepsis, has not been extensively investigated for its therapeutic potential in bacterial meningitis. This study aims to assess the neuroprotective effects of ulinastatin on bacterial meningitis and elucidate its underlying mechanism. The rat model of bacterial meningitis was established by intracerebral injection of Escherichia coli. 3-week-old SD rats were randomly divided into 5 groups with 8 rats in each group, including control group, E.coli group, E.coli + UTI group (ulinastatin 50000IU/kg), E.coli + UTI + PMA group (ulinastatin 50000IU/kg + PMA 200 ug/kg), and E.coli + PMA group(PMA 200 ug/kg). Behavioral changes were assessed by Loeffler neurobehavioral score. Histomorphologic changes and apoptosis were assessed by hematoxylin and eosin staining, Nissl staining and TUNEL staining. Immunohistochemistry and immunofluorescence and western blotting were used to detect the expression levels of zonula occludens-1 (ZO-1) and phosphorylation protein kinase C (PKCα).It was found that ulinastatin treatment in Escherichia coli meningitis rats improved neurological function, alleviated meningeal inflammatory infiltration, reduced neuronal death, promoted the integrity of the blood-brain barrier structure. Moreover, phorbol myristate acetate (PMA, a protein kinase C activator), blocked the effective action of ulinastatin. These findings suggest that ulinastatin had neuroprotective effects on bacterial meningitis by inhibiting PKCα phosphorylation and reducing ZO-1 degradation, demonstrating that ulinastatin may be a promising strategy in the treatment of bacterial meningitis.

Deletion of protein kinase C θ attenuates hepatic ischemia/reperfusion injury and further elucidates its mechanism in pathophysiology.

Objectives: Hepatic ischemia-reperfusion (HIR) is a severe process in pathophysiology that occurs clinically in hepatectomy, and hepatic transplantations. The present study aimed to investigate the effect of PKC θ deletion against HIR injury and elucidate its mechanism in pathophysiology. Materials and Methods: HIR injury was induced in wild-type and PKC θ deletion mice treated with or without heme. The ALT and AST levels were determined to evaluate liver function. HIR injury was observed via histological examination. Oxidative stress and inflammatory response markers, and their signaling pathways were detected. Results: The study found that PKC θ knockout decreased serum AST and ALT levels when compared to the WT mice. Furthermore, heme treatment significantly reduced the ALT and AST levels of the PKC θ deletion mice compared with the untreated PKC θ deletion mice. PKC θ deletion markedly elevated superoxide dismutase activity in the liver tissue, reduced malondialdehyde content in the tissue, and the serum TNF-α and IL-6 levels compared with the WT mice. Heme treatment was observed to elevate the activity of SOD and reduced MDA content and serum of TNF-α and IL 6 in the PKC θ deletion animals. Meanwhile, heme treatment increased HO-1 and Nrf 2 protein expression, and reduced the levels of TLR4, phosphorylated NF-κB, and IKB-α. Conclusion: These findings suggested that PKC θ deletion ameliorates HIR, and heme treatment further improves HIR, which is related to regulation of PKC θ deletion on Nrf 2/HO-1 and TLR4/NF-κB/IKB α pathway.

Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species­mediated MMP­9 activation and invasion in MCF­7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF­7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)­9 were assessed by reverse transcription­quantitative PCR and western blotting, and the activity of MMP­9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced MMP­9 expression and invasion of MCF­7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA­induced MMP­9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA­induced MMP­9 expression and invasion in MCF­7 cells. NOX1­ and NOX5­specific small interfering RNAs attenuated TPA­induced MMP­9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA­induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA­induced intracellular ROS levels, MMP­9 expression and NOX activity in MCF­7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA­induced MMP­9 expression and invasion of MCF­7 breast cancer cells. Furthermore, the present study indicated that TPA­induced MMP­9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti­invasive effects in breast cancer.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Endogenous hydrogen peroxide positively regulates secretion of a gut-derived peptide in neuroendocrine potentiation of the oxidative stress response in C. elegans.

The gut-brain axis mediates bidirectional signaling between the intestine and the nervous system and is critical for organism-wide homeostasis. Here we report the identification of a peptidergic endocrine circuit in which bidirectional signaling between neurons and the intestine potentiates the activation of the antioxidant response in C. elegans in the intestine. We identify a FMRF-amide-like peptide, FLP-2, whose release from the intestine is necessary and sufficient to activate the intestinal oxidative stress response by promoting the release of the antioxidant FLP-1 neuropeptide from neurons. FLP-2 secretion from the intestine is positively regulated by endogenous hydrogen peroxide (H2O2) produced in the mitochondrial matrix by sod-3/superoxide dismutase, and is negatively regulated by prdx-2/peroxiredoxin, which depletes H2O2 in both the mitochondria and cytosol. H2O2 promotes FLP-2 secretion through the DAG and calciumdependent protein kinase C family member pkc-2 and by the SNAP25 family member aex-4 in the intestine. Together, our data demonstrate a role for intestinal H2O2 in promoting inter-tissue antioxidant signaling through regulated neuropeptide-like protein exocytosis in a gut-brain axis to activate the oxidative stress response.

Sclerostin inhibits Protein kinase C inhibitor GÖ6976 induced osteogenic differentiation of dental follicle cells.

Human dental follicle cells (DFCs) as multipotent stem cells are currently investigated within the field of regenerative medicine considering their potential for the regeneration of dental tissues, bone defects caused by periodontal or degenerative diseases and the treatment of craniofacial disorders. However, molecular mechanisms of the differentiation into mineralizing cells are still inadequately understood. Previous studies have shown that GÖ6976, an inhibitor of classical isoforms of protein kinase C (PKC), enhanced ostogenic differentiation of DFCs. A possible mechanism for increased osteogenic differentiation could be the regulation of ossification inhibitors. This study therefore investigated whether the osteogenic differentiation inhibitor sclerostin (SOST) is regulated by GÖ6976 and whether the addition of sclerostin attenuates the stimulating effect of the PKC inhibitor. We demonstrated that the expression of the sclerostin gene decreased after PKC inhibition by GÖ6976 and that its gene expression is likely maintained by PKC via the BMP signaling pathway. Furthermore, supplementation of osteogenic differentiation medium with sclerostin impairs GÖ6976-induced differentiation of DFCs. Our data suggest that regulation of sclerostin mediates PKC inhibition-induced mineralization of DFCs.

Disclosing the Novel Protective Mechanisms of Ocrelizumab in Multiple Sclerosis: The Role of PKC Beta and Its Down-Stream Targets.

Ocrelizumab (OCR) is a humanized anti-CD20 monoclonal antibody approved for both Relapsing and Primary Progressive forms of Multiple Sclerosis (MS) treatment. OCR is postulated to act via rapid B cell depletion; however, by analogy with other anti-CD20 agents, additional effects can be envisaged, such as on Protein Kinase C (PKC). Hence, this work aims to explore novel potential mechanisms of action of OCR in peripheral blood mononuclear cells from MS patients before and after 12 months of OCR treatment. We first assessed, up-stream, PKCßII and subsequently explored two down-stream pathways: hypoxia-inducible factor 1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF), and human antigen R (HuR)/manganese-dependent superoxide dismutase (MnSOD) and heat shock proteins 70 (HSP70). At baseline, higher levels of PKCßII, HIF-1α, and VEGF were found in MS patients compared to healthy controls (HC); interestingly, the overexpression of this inflammatory cascade was counteracted by OCR treatment. Conversely, at baseline, the content of HuR, MnSOD, and HSP70 was significantly lower in MS patients compared to HC, while OCR administration induced the up-regulation of these neuroprotective pathways. These results enable us to disclose the dual positive action of OCR: anti-inflammatory and neuroprotective. Therefore, in addition to B cell depletion, the effect of OCR on these molecular cascades can contribute to counteracting disease progression.

Diacylglycerol O-acyltransferase 1 inhibitor increases plasma alanine aminotransferase and aspartate aminotransferase activities via a shedding of the intestinal villi and an increase in intestinal permeability in rats.

Diacylglycerol O-acyltransferase 1 (DGAT1) is a key enzyme for fat absorption step in the enterocytes. We previously reported that DGAT1 inhibition increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in corn oil-loaded rats via protein kinase C (PKC) activation. In the present study, we investigated the mechanism with respect to the morphology and permeability of the small intestine, focusing on PKC function, and found that shortening of the intestinal villi and a decrease in the number of tdT-mediated dUTP-biotin nick-end labeling-positive cells in the tips of the villi were observed in the jejunum of DGAT1 inhibitor-treated rats loaded with corn oil. These results suggested that the tips of the villi were shed into the intestinal lumen. Next, fluorescein isothiocyanate-dextran, 110 kDa (FD-110) was administered intraduodenally to DGAT1 inhibitor-treated rats loaded with corn oil and we found that plasma FD-110 concentrations increased, indicating that the intestinal permeability to molecules with a molecular weight of approximately 110,000 (e.g., ALT and AST) increased. Taken together, the present results suggested that DGAT1 inhibitor-treatment in combination with corn oil causes ALT and AST to leak from the enterocytes into the blood by shedding the tips of the intestinal villi and increasing intestinal permeability.

Cells and circuits for amygdala neuroplasticity in the transition to chronic pain.

Maladaptive plasticity is linked to the chronification of diseases such as pain, but the transition from acute to chronic pain is not well understood mechanistically. Neuroplasticity in the central nucleus of the amygdala (CeA) has emerged as a mechanism for sensory and emotional-affective aspects of injury-induced pain, although evidence comes from studies conducted almost exclusively in acute pain conditions and agnostic to cell type specificity. Here, we report time-dependent changes in genetically distinct and projection-specific CeA neurons in neuropathic pain. Hyperexcitability of CRF projection neurons and synaptic plasticity of parabrachial (PB) input at the acute stage shifted to hyperexcitability without synaptic plasticity in non-CRF neurons at the chronic phase. Accordingly, chemogenetic inhibition of the PB→CeA pathway mitigated pain-related behaviors in acute, but not chronic, neuropathic pain. Cell-type-specific temporal changes in neuroplasticity provide neurobiological evidence for the clinical observation that chronic pain is not simply the prolonged persistence of acute pain.

Cleavage of protein kinase c δ by caspase-3 mediates proinflammatory cytokine-induced apoptosis in pancreatic islets.

In type 1 diabetes (T1D), autoreactive immune cells infiltrate the pancreas and secrete proinflammatory cytokines that initiate cell death in insulin producing islet ß-cells. Protein kinase C δ (PKCδ) plays a role in mediating cytokine-induced ß-cell death; however, the exact mechanisms are not well understood. To address this, we used an inducible ß-cell specific PKCδ KO mouse as well as a small peptide inhibitor of PKCδ. We identified a role for PKCδ in mediating cytokine-induced ß-cell death and have shown that inhibiting PKCδ protects pancreatic ß-cells from cytokine-induced apoptosis in both mouse and human islets. We determined that cytokines induced nuclear translocation and activity of PKCδ and that caspase-3 cleavage of PKCδ may be required for cytokine-mediated islet apoptosis. Further, cytokine activated PKCδ increases activity both of proapoptotic Bax with acute treatment and C-Jun N-terminal kinase with prolonged treatment. Overall, our results suggest that PKCδ mediates cytokine-induced apoptosis via nuclear translocation, cleavage by caspase-3, and upregulation of proapoptotic signaling in pancreatic ß-cells. Combined with the protective effects of PKCδ inhibition with δV1-1, the results of this study will aid in the development of novel therapies to prevent or delay ß-cell death and preserve ß-cell function in T1D.

Effect of phenolic-hydroxy-group incorporation on the biological activity of a simplified aplysiatoxin analog with an (R)-(-)-carvone-based core.

We synthesized a phenolic hydroxy group-bearing version (1) of a simplified analog of aplysiatoxin comprising a carvone-based conformation-controlling unit. Thereafter, we evaluated its antiproliferative activity against human cancer cell lines and its binding affinity to protein kinase C (PKC) isozymes. The antiproliferative activity and PKC-binding ability increased with the introduction of the phenolic hydroxy group. The results of molecular dynamics simulations and subsequent relative binding free-energy calculations conducted using an alchemical transformation procedure showed that the phenolic hydroxy group in 1 could form a hydrogen bond with a phospholipid and the PKC. The former hydrogen bonding formation facilitated the partitioning of the compound from water to the phospholipid membrane and the latter compensated for the loss of hydrogen bond with the phospholipid upon binding to the PKC. This information may facilitate the development of rational design methods for PKC ligands with additional hydrogen bonding groups.