Protein Kinase C Activity Research Articles

A high-concentrate diet provokes inflammation, endoplasmic reticulum stress, and apoptosis in mammary tissue of dairy cows through the upregulation of STIM1/ORAI1

High-concentrate feeding can induce subacute ruminal acidosis, which leads to mammary tissue injury in dairy cows. Therefore, the purpose of this research was to evaluate the effect of high-concentrate feeding on STIM1 (stromal interaction molecule 1)/ORAI1 (Orai calcium release-activated calcium modulator 1)-mediated inflammation, endoplasmic reticulum stress (ERS), and apoptosis in the mammary tissue of dairy cows. A total of 12 healthy mid-lactating Holstein cows of similar weight were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 6:4) and a low-concentrate (LC) group (concentrate:forage = 4:6). The trial lasted for 3 wk. After the feeding experiment, rumen fluid, lacteal vein blood, and mammary tissue samples were collected. The results showed that the HC diet significantly increased blood lipopolysaccharide levels, decreased ruminal pH, and upregulated the concentrations of Ca2+ and proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, and the enzyme activities of caspase-3, caspase-9, PKC, and IKK. The upregulation of STIM1, ORAI1, PKCα, IKKβ, phosphorylated-IκBα, phosphorylated-p65, TNF-α, and IL-1α proteins in the HC group indicated activation of the STIM1/ORAI1-mediated inflammatory signaling pathway compared with that in the LC group. The HC diet also induced ERS by increasing the mRNA and protein abundances of GRP78, CHOP, PERK, ATF6, and IRE1α in the mammary tissue. Compared with the LC group, the mRNA expression levels and protein abundances of caspase-3, cleaved caspase-3, caspase-9, and BAX were markedly increased in the HC group. However, the mRNA and protein expression levels of Bcl-2 were significantly decreased in the HC group. Therefore, this study demonstrated that the HC diet can activate the store-operated calcium entry channel by upregulating the expression of STIM1 and ORAI1 and induce inflammation, ERS, and apoptosis in the mammary tissue of dairy cows.

Acyl-CoA:diacylglycerol acyltransferase 1 inhibition in the small intestine increases plasma transaminase activity via the activation of protein kinase C pathway.

Acyl-CoAdiacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the fat absorption step in enterocytes. We previously reported that the pharmacological inhibition of DGAT1 increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in corn oil-loaded rats without any sign of hepatotoxicity. In this study, we investigated this mechanism. We found that this elevation occurred only during the pharmacologically active period of a DGAT1 inhibitor and the magnitude did not depend on the volume of corn oil. In addition, this elevation was not accompanied by increases in ALT or AST mRNA levels in the small intestine and liver. To clarify a lipid component responsible for this elevation, rats were treated with free fatty acids instead of corn oil and no plasma ALT elevation was observed. Next, rats were pretreated with inhibitors of monoacylglycerol acyltransferase 2 and intestinal microsomal triglyceride transfer protein instead of the DGAT1 inhibitor, but no plasma ALT elevation was observed after corn oil loading. Since the results suggested a possible role of diacylglycerol (DAG), which activates protein kinase C (PKC), we measured PKC activity in the small intestine and found that the activity was increased by treatment with the DGAT1 inhibitor and corn oil. Moreover, rats pretreated with a PKC inhibitor in combination with the DGAT1 inhibitor showed suppression of plasma ALT elevation. Taken together, the present results suggest that DAG accumulation induced by pharmacological DGAT1 inhibition and resultant PKC activation in enterocytes are involved in the increase in plasma ALT and AST activity in rats.

Roles of c-Fos, EGR-1, PKA, and PKC in cognitive dysfunction in rats after propofol anesthesia

This study aimed to investigate possible changes in the spatial memory of rats and the expression or activity of EGR-1, c-Fos, PKA, and PKC after propofol anesthesia. Thirty-six Sprague-Dawley rats aged 20 months and 36 Sprague-Dawley rats aged three months were each randomly divided into three groups: the control group, the Morris Water Maze (MWM) group, and the propofol group. In the propofol groups of both young and aged rats, the rats were anesthetized by propofol for two or four hours and then performed the MWM test two days or two weeks after anesthesia to assess cognitive function. EGR-1, c-Fos, PKA, and PKC expressions in the rat hippocampus were determined via immunohistochemistry. For the older rats, the escape latency in the P4h/2d group was significantly prolonged (P < 0.05), and the learning curve was right-shifted in the P4h/2w group (P < 0.05). The expression levels of EGR-1, c-Fos, PKA, and PKC in the MWM groups were significantly higher than those in the control groups (P < 0.05). In the P4h/2d group of aged rats, the expression levels of both PKA and PKC were decreased compared with those of the MWM groups. The decreased expression of both protein kinases may be responsible for the observed impairment after propofol anesthesia.

Antidepressant-like effect of caffeic acid: Involvement of the cellular signaling pathways

Caffeic acid is a phenolic compound widely distributed in plants and beverages such as coffee. Although its mechanism of action is poorly understood, caffeic acid reportedly induces antidepressant-like and neuroprotective effects. This study aimed to investigate the involvement of cellular signaling pathways in acute antidepressant-like effect induced by caffeic acid in mice. All procedures were approved by the Institutional Animal Ethics Committee of the UNIVALI n. 021/2013. Female Swiss mice were administered with vehicle, caffeic acid (5 mg/ kg, p.o.), inhibitor (H-89, U0126, chelerythrine, or PD9859, i.c.v.) or caffeic acid plus inhibitor. The behavioral effects were evaluated 1h after the administration of compounds to mice using tail suspension test (TST) and open field test (OFT). The results showed that the antidepressant- like effect of caffeic acid in mice was possibly mediated by the activation of PKA, MEK 1/2, PKC and MAPK (as assessed using TST), without compromising their locomotor activity (as assessed using OFT). Our results demonstrated, at least in part, the pathways involved in the neuroprotective and behavioral effects of caffeic acid.

Insulin-like Growth Factor II Prevents MPP+ and Glucocorticoid Mitochondrial-Oxidative and Neuronal Damage in Dopaminergic Neurons.

Stress seems to contribute to Parkinson’s disease (PD) neuropathology, probably by dysregulation of the hypothalamic–pituitary–adrenal axis. Key factors in this pathophysiology are oxidative stress and mitochondrial dysfunction and neuronal glucocorticoid-induced toxicity. The insulin-like growth factor II (IGF-II), a pleiotropic hormone, has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Our aim was to examine the protective effect of IGF-II on a dopaminergic cellular combined model of PD and mild to moderate stress measuring oxidative stress parameters, mitochondrial and neuronal markers, and signalling pathways. IGF-II counteracts the mitochondrial-oxidative damage produced by the toxic synergistic effect of corticosterone and 1-methyl-4-phenylpyridinium, protecting dopaminergic neurons from death and neurodegeneration. IGF-II promotes PKC activation and nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. Thus, IGF-II is a potential therapeutic tool for treatment and prevention of disease progression in PD patients suffering mild to moderate emotional stress.

MicroRNA 630 Represses NANOG Expression through Transcriptional and Post-Transcriptional Regulation in Human Embryonal Carcinoma Cells.

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3′UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3′UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3′UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3′UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3′UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3′UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

Magnesium and Morphine in the Treatment of Chronic Neuropathic Pain-A Biomedical Mechanism of Action.

The effectiveness of opioids in the treatment of neuropathic pain is limited. It was demonstrated that magnesium ions (Mg2+), physiological antagonists of N-methyl-D-aspartate receptor (NMDAR), increase opioid analgesia in chronic pain. Our study aimed to determine the molecular mechanism of this action. Early data indicate the cross-regulation of µ opioid receptor (MOR) and NMDAR in pain control. Morphine acting on MOR stimulates protein kinase C (PKC), while induction of NMDAR recruits protein kinase A (PKA), leading to a disruption of the MOR-NMDAR complex and promoting functional changes in receptors. The mechanical Randall-Selitto test was used to assess the effect of chronic Mg2+ and morphine cotreatment on streptozotocin-induced hyperalgesia in Wistar rats. The level of phosphorylated NMDAR NR1 subunit (pNR1) and phosphorylated MOR (pMOR) in the periaqueductal gray matter was determined with the Western blot method. The activity of PKA and PKC was examined by standard enzyme immunoassays. The experiments showed a reduction in hyperalgesia after coadministration of morphine (5 mg/kg intraperitoneally) and Mg2+ (40 mg/kg intraperitoneally). Mg2+ administered alone significantly decreased the level of pNR1, pMOR, and activity of both tested kinases. The results suggest that blocking NMDAR signaling by Mg2+ restores the MOR-NMDAR complex and thus enables morphine analgesia in neuropathic rats.

Muscarinic signaling regulates voltage-gated potassium channel KCNQ2 phosphorylation in the nucleus accumbens via protein kinase C for aversive learning.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

Wogonin and Alleviation of Hyperglycemia via Inhibition of DAG Mediated PKC Expression. A Brief Insight.

Protein kinase C (PKC) is a family of protein kinase enzymes that can phosphorylate other proteins and influence their functions, such as signal transduction, cell survival, and death. Increased diacylglycerol (DAG) concentrations, which are typically observed raised in hyperglycemic situations such as diabetes mellitus, can also activate PKC enzymes (DM). On the other hand, PKC isomers have been shown to play an essential role in diabetes and many hyperglycemic complications, most importantly atherosclerosis and diabetic cardiomyopathy (DCM). As a result, blocking PKC activation via DAG can prevent hyperglycemia and related consequences, such as DCM. Wogonin is a herbal medicine which has anti-inflammatory properties, and investigations show that it scavenge oxidative radicals, attenuate nuclear factor-kappa B (NF-κB) activity, inhibit several essential cell cycle regulatory genes, block nitric oxide (NO) and suppress cyclooxygenase- 2 (COX-2). Furthermore, several investigations show that wogonin also attenuates diacylglycerol DAG levels in diabetic mice. Since the DAG-PKC pathway is linked with hyperglycemia and its complications, Wogonin-mediated DAG-PKC attenuation can help treat hyperglycemia and its complications.

Alpha-mangostin dephosphorylates ERM to induce adhesion and decrease surface stiffness in KG-1 cells.

Surface stiffness is a unique indicator of various cellular states and events and needs to be tightly controlled. α-Mangostin, a natural compound with numerous bioactivities, reduces the mechanical stiffness of various cells; however, the mechanism by which it affects the actin cytoskeleton remains unclear. We aimed to elucidate the mechanism underlying α-mangostin activity on the surface stiffness of leukocytes. We treated spherical non-adherent myelomonocytic KG-1 cells with α-mangostin; it clearly reduced their surface stiffness and disrupted their microvilli. The α-mangostin-induced reduction in surface stiffness was inhibited by calyculin A, a protein phosphatase inhibitor. α-Mangostin also induced KG-1 cell adhesion to a fibronectin-coated surface. In KG-1 cells, a decrease in surface stiffness and the induction of cell adhesion are largely attributed to the dephosphorylation of ezrin/radixin/moesin proteins (ERMs); α-mangostin reduced the levels of phosphorylated ERMs. It further increased protein kinase C (PKC) activity. α-Mangostin-induced KG-1 cell adhesion and cell surface softness were inhibited by the PKC inhibitor GF109203X. The results of the present study suggest that α-mangostin decreases stiffness and induces adhesion of KG-1 cells via PKC activation and ERM dephosphorylation.

Abstract 13450: Role of PKC in Metabolic Regulation of Endothelial SK Channels

Introduction: Diabetes is associated with increases in metabolites NADH, and PKC. NADH has been recently found to inactivate endothelial SK channels. 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 NADH dysregulation of endothelial SK channels. Hypothesis: We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels and coronary relaxation function. Methods: SK channel 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, or PKC inhibitors or endothelial PKC α /PKC β knock-down by using short interfering RNA. The in-vitro coronary arterioles (n = 5) from the harvested atrial tissue samples of patients undergoing cardiac surgery were perfused with NADH in the presence or absence of PKC β inhibitor LY333531. Vascular reactivity was measured by vessel myography in the presence of vasoconstrictor and vasodilators. Results: NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p&lt;0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC β inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p&gt;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, PKC β knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p&gt;0.05, vs. control, Fig. G). Incubation of vessels with NADH significantly reduced relaxation response to the SK activator NS309 and this effect was abolished in the presence of LY333531. Conclusions: The metabolite NADH dysregulation of endothelial SK channels and impairment of endothelium-dependent relaxation function was via the PKC β signaling pathway.

EFFECT OF SUPERCRITICAL FLUID EXTRACT OF ZINGIBER OFFICINALIS IN THE MANAGEMENT OF DIABETES AND ITS RELATED COMPLICATIONS

Diabetes mellitus is referred as diabetes which is the metabolic disorder. Major symptoms of diabetes include increased urination &amp; thrust, head ache, high degree of sudden weight loss. Due to increase in blood glucose levels it causes trigger of various pathways which ultimately leads to various diabetic complications. Pathways like which includes Polyol Pathway, Hexosamine Pathway, Activation of PK-C (Protein Kinase-C), &amp; formation of Advance Glycation End Products. For the diagnosis of diabetes two major tests are done blood glucose level and Glycated hemoglobin content (HbA1c). Ayurveda is the super bank for various molecules to treat any disease. Here we have chosen the super critical fluid extract of Ginger that is Zingiber Officinale. In our study we have used Streptozotocin induced diabetes mellitus model by using rats. Our result showed promising effects in the streptozotocine induced diabetes and immunomodulatory activity was also shown by the extract. Overall in conclusion it Zingiber Officinale supercritical fluid extract can be considered as source of multitalented molecules which can be proven better for the management of diabetes and its related

EFFECT OF SUPERCRITICAL FLUID EXTRACT OF ZINGIBER OFFICINALIS IN THE MANAGEMENT OF DIABETES AND ITS RELATED COMPLICATIONS

Diabetes mellitus is referred as diabetes which is the metabolic disorder. Major symptoms of diabetes include increased urination &amp; thrust, head ache, high degree of sudden weight loss. Due to increase in blood glucose levels it causes trigger of various pathways which ultimately leads to various diabetic complications. Pathways like which includes Polyol Pathway, Hexosamine Pathway, Activation of PK-C (Protein Kinase-C), &amp; formation of Advance Glycation End Products. For the diagnosis of diabetes two major tests are done blood glucose level and Glycated hemoglobin content (HbA1c). Ayurveda is the super bank for various molecules to treat any disease. Here we have chosen the super critical fluid extract of Ginger that is Zingiber Officinale. In our study we have used Streptozotocin induced diabetes mellitus model by using rats. Our result showed promising effects in the streptozotocine induced diabetes and immunomodulatory activity was also shown by the extract. Overall in conclusion it Zingiber Officinale supercritical fluid extract can be considered as source of multitalented molecules which can be proven better for the management of diabetes and its related

Hydrogen peroxide and phosphoinositide metabolites synergistically regulate a cation current to influence neuroendocrine cell bursting.

In various neurons, including neuroendocrine cells, non-selective cation channels elicit plateau potentials and persistent firing. Reproduction in the marine snail Aplysia californica is initiated when the neuroendocrine bag cell neurons undergo an afterdischarge, that is, a prolonged period of enhanced excitability and spiking during which egg-laying hormone is released into the blood. The afterdischarge is associated with both the production of hydrogen peroxide (H2 O2 ) and activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3 ). We previously demonstrated that H2 O2 gates a voltage-dependent cation current and evokes spiking in bag cell neurons. The present study tests if DAG and IP3 impact the H2 O2 -induced current and excitability. In whole-cell voltage-clamped cultured bag cell neurons, bath-application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the H2 O2 -induced current, which was amplified by the inclusion of IP3 in the pipette. A similar outcome was produced by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. In current-clamp, OAG or OAG plus IP3 , elevated the frequency of H2 O2 -induced bursting. PKC is also triggered during the afterdischarge; when PKC was stimulated with phorbol 12-myristate 13-acetate, it caused a voltage-dependent inward current with a reversal potential similar to the H2 O2 -induced current. Furthermore, PKC activation followed by H2 O2 reduced the onset latency and increased the duration of action potential firing. Finally, inhibiting nicotinamide adenine dinucleotide phosphate oxidase with 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine diminished evoked bursting in isolated bag cell neuron clusters. These results suggest that reactive oxygen species and phosphoinostide metabolites may synergize and contribute to reproductive behaviour by promoting neuroendocrine cell firing. KEY POINTS: Aplysia bag cell neurons secrete reproductive hormone during a lengthy burst of action potentials, known as the afterdischarge. During the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol trisphosphate (IP3 ). Subsequent activation of protein kinase C (PKC) leads to H2 O2 production. H2 O2 evokes a voltage-dependent inward current and action potential firing. Both a DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and IP3 enhance the H2 O2 -induced current, which is mimicked by the PLC activator, N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide. The frequency of H2 O2 -evoked afterdischarge-like bursting is augmented by OAG or OAG plus IP3 . Stimulating PKC with phorbol 12-myristate 13-acetate shortens the latency and increases the duration of H2 O2 -induced bursts. The nicotinamide adenine dinucleotide phosphate oxidase inhibitor, 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine, attenuates burst firing in bag cell neuron clusters.

Siglec-15 Is a Novel Immunomodulatory Protein and Therapeutic Target in Acute Lymphoblastic Leukemia

Immunotherapies have recently shown efficacy in treatment of aggressive, refractory pediatric B cell acute lymphoblastic leukemia (B-ALL), which remains one of the leading causes of cancer-related death in children. The immune evasion mechanisms of B-ALL are still being explored to discover new therapeutic targets and improve patient outcomes.Recent reports have implicated a role for the molecule Siglec-15 (Sig15) in regulating immune response in solid tumor-infiltrating macrophages. Our lab has found higher expression of SIGLEC15 at the RNA level in primary pediatric B-ALL as compared to healthy donor controls, as well as at the RNA and protein levels across a panel of B-ALL, T cell acute lymphoblastic leukemia (T-ALL), and diffuse large B cell lymphoma (DLBCL) cell lines compared to healthy donor PBMCs. Higher expression of SIGLEC15 in pediatric B-ALL samples from the TARGET database correlates with markers of PKC and NFκB activation known to drive B-ALL leukemogenesis, which we have demonstrated to regulate Sig15 RNA and protein expression in vitro.Knockout of Siglec15 expression in a BCR-ABL1 + murine model of B-ALL engrafted in immunocompetent and Rag1 -/- immunodeficient recipients resulted in leukemia clearance in immunocompetent, but not immunodeficient, recipients and 100% survival (Figure A, p=0.01 Sig15 KO into WT vs. Rag1 -/-). Further study indicates that Siglec15 expression on these leukemia cells suppresses T cell effector and memory population expansion at 7 days post-engraftment (Figure B) and correlates with higher levels of IL-10 and lower levels of CCL17 present in the bone marrow, representing a more immunosuppressive bone marrow milieu. These data suggest a prominent role for Sig15 in the suppression of adaptive immune response to B-ALL as well as other hematological malignancies.We have also reported for the first time the release of a soluble form of Sig15 (sSig15), which we have demonstrated to circulate at higher levels in the plasma of pediatric B-ALL patients compared to healthy donors (Figure C, ****P≤0.0001). Detection of this sSig15 negatively correlated with circulating levels of IL-12 and IL-1α/β (Figure D, depicting correlations of cytokines using Pearson's r), suggesting sSig15 levels correspond to a systemically immunosuppressive phenotype. Flow cytometry of fresh pediatric B ALL cells demonstrates expression of surface Sig15 in a subset of cases. Thus, Sig15 has the capacity to promote immunosuppressive effects at both marrow-localized and systemic levels.Together, these results suggest Siglec-15 is a novel, potent immunosuppressive molecule active in leukemia progression that may be targeted therapeutically to activate T lymphocytes against leukemia cells. [Display omitted] DisclosuresAbukharma: NextCure Inc.: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company.

Induction of ß Cell Replication by Small Molecule-Mediated Menin Inhibition and Combined PKC Activation and TGF‑ß Inhibition as Revealed by A Refined Primary Culture Screening.

Objective Pancreatic β cells are recognized as central players in the pathogenesis of types 1 and 2 diabetes. Efficient and robust primary culture methods are required to interrogate β cell biology and screen potential anti-diabetic therapeutics. The aim of this study was to refine monolayer culture of beta cells and to investigate potential inducers of beta cell proliferation. Materials and Methods In this experimental study, we compared different culture methods to optimize conditions required for a monolayer culture of rat pancreatic islet cells in order to facilitate image analysis-based assays. We also used the refined culture method to screen a group of rationally selected candidate small molecules and their combinations to determine their potential proliferative effects on the β cells. Results Ham’s F10 medium supplemented with 2% foetal bovine serum (FBS) in the absence of any surface coating provided a superior monolayer β cell culture, while other conditions induced fibroblast-like cell growth or multilayer cell aggregation over two weeks. Evaluation of candidate small molecules showed that a menin inhibitor MI-2 and a combination of transforming growth factor-β (TGF-β) inhibitor SB481542 and protein kinase C (PKC) activator indolactam V (IndV) significantly induced replication of pancreatic β cells. ConclusionOverall, our optimized culture condition provided a convenient approach to study the cultured pancreatic islet cells and enabled us to detect the proliferative effect of menin inhibition and combined TGF-β inhibition and PKC activation, which could be considered as potential strategies for inducing β cell proliferation and regeneration.

Exercise prevents HFD-induced insulin resistance risk: involvement of TNF-\u03b1 level regulated by vagus nerve-related anti-inflammatory pathway in the spleen

ObjectivesRegular physical exercise can improve insulin resistance in insulin target tissues. However, the mechanisms about the beneficial effect of exercise on insulin resistance are not yet fully resolved. This study was carried out to address whether insulin resistance improvement by exercise is involved in an anti-inflammatory pathway in the spleen in high-fat diet (HFD) feeding mice.MethodsMale C57Bl/6J mice with or without subdiaphragmatic vagotomy (sVNS) were subjected to medium-intensity treadmill exercise during HFD feeding. Glucose tolerance test and insulin tolerance test were detected, and spleen acetylcholine level, choline acetyltransferase activity (ChAT), protein kinase C (PKC) and tumor necrosis factor-alpha (TNF-α) were assayed.ResultsWe found that exercise significantly improves HFD-induced glucose intolerance and insulin resistance, along with an increase in acetylcholine level, ChAT activity, and PKC activity, and decrease in TNF-α level in the system and the spleen from HFD-fed mice. However, sVNS abolished the beneficial effect of exercise on glucose intolerance and insulin resistance, decreased acetylcholine level, ChAT activity, and PKC activity, and increase TNF-α level of the spleen in HFD-mice exercise intervention.ConclusionsThese data reveal that the prevention of HFD-associated insulin resistance by exercise intervention involves reducing splenic TNF-α level, which is mediated by cholinergic anti-inflammatory activity via influencing PKC activity, ChAT activity, and acetylcholine concentration in mice spleen.

Protein Kinase C-Dependent Effects of Neurosteroids on Synaptic GABAA Receptor Inhibition Require the δ-Subunit.

The dorsal motor nucleus of the vagus (DMV) contains preganglionic motor neurons important for interpreting sensory input from the periphery, integrating that information, and coding the appropriate parasympathetic (vagal) output to target organs. Despite the critical role of hormonal regulation of vagal motor output, few studies examine the role of neurosteroids in the regulation of the DMV. Of the few examinations, no studies have investigated the potential impact of allopregnanolone (Allo), a neuroactive progesterone-derivative, in the regulation of neurotransmission on the DMV. Since DMV neuronal function is tightly regulated by GABAA receptor activity and Allo is an endogenous GABAA receptor ligand, the present study used in vitro whole cell patch clamp to investigate whether Allo alters GABAergic neurotransmission to DMV neurons. Although Allo did not influence GABAergic neurotransmission during initial application (5–20 min), a TTX-insensitive prolongment of decay time and increase in frequency of GABAergic currents was established after Allo was removed from the bath for at least 30 min (LtAllo). Inhibition of protein kinase C (PKC) abolished these effects, suggesting that PKC is largely required to mediate Allo-induced inhibition of the DMV. Using mice that lack the δ-subunit of the GABAA receptor, we further confirmed that PKC-dependent activity of LtAllo required this subunit. Allo also potentiated GABAA receptor activity after a repeated application of δ-subunit agonist, suggesting that the presence of Allo encodes stronger δ-subunit-mediated inhibition over time. Using current clamp recording, we demonstrated that LtAllo-induced inhibition is sufficient to decrease action potential firing and excitability within DMV neurons. We conclude that the effects of LtAllo on GABAergic inhibition are dependent on δ-subunit and PKC activation. Taken together, DMV neurons can undergo long lasting Allo-dependent GABAA receptor plasticity.

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels.

Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.