Intracellular PKC Research Articles

Roles of metabotropic glutamate receptor 5 in low [Mg2+]o-induced interictal epileptiform activity in rat hippocampal slices.

Group I metabotropic glutamate receptors (mGluRs) modulate postsynaptic neuronal excitability and epileptogenesis. We investigated roles of group I mGluRs on low extracellular Mg2+ concentration ([Mg2+]o)-induced epileptiform activity and neuronal cell death in the CA1 regions of isolated rat hippocampal slices without the entorhinal cortex using extracellular recording and propidium iodide staining. Exposure to Mg2+-free artificial cerebrospinal fluid can induce interictal epileptiform activity in the CA1 regions of rat hippocampal slices. MPEP, a mGluR 5 antagonist, significantly inhibited the spike firing of the low [Mg2+]o-induced epileptiform activity, whereas LY367385, a mGluR1 antagonist, did not. DHPG, a group 1 mGluR agonist, significantly increased the spike firing of the epileptiform activity. U73122, a PLC inhibitor, inhibited the spike firing. Thapsigargin, an ER Ca2+-ATPase antagonist, significantly inhibited the spike firing and amplitude of the epileptiform activity. Both the IP3 receptor antagonist 2-APB and the ryanodine receptor antagonist dantrolene significantly inhibited the spike firing. The PKC inhibitors such as chelerythrine and GF109203X, significantly increased the spike firing. Flufenamic acid, a relatively specific TRPC 1, 4, 5 channel antagonist, significantly inhibited the spike firing, whereas SKF96365, a relatively non-specific TRPC channel antagonist, did not. MPEP significantly decreased low [Mg2+]o DMEM-induced neuronal cell death in the CA1 regions, but LY367385 did not. We suggest that mGluR 5 is involved in low [Mg2+]oinduced interictal epileptiform activity in the CA1 regions of rat hippocampal slices through PLC, release of Ca2+ from intracellular stores and PKC and TRPC channels, which could be involved in neuronal cell death.

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

EXTH-31. EXPLOITING THE GENETIC DEPENDENCY ON PI3K/MTOR SIGNALING FOR THE TREATMENT OF H3-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract Recurring activating mutations, amplifications and allelic loss of the negative repressors of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling genes are overarching contributors to the poor survival of patients with H3K27-altered diffuse midline gliomas (DMG) diagnosed in the pons (diffuse intrinsic pontine glioma – DIPG), highlighting the clinical potential of therapies that target this axis. Employing a loss-of-function CRISPR-Cas9 gene deletion screen, we identified PIK3CA and MTOR as survival dependences in DMG/DIPG patient derived cell line models (n=36), highlighting the clinical potential of targeting PI3K/Akt/mTOR. The brain-penetrant, PI3K/mTOR inhibitor paxalisib is currently under clinical investigation in DIPG (NCT03696355 and NCT05009992), but dosing limiting toxicities including hyperglycaemia, mucositis and skin irritation limit patient compliance. To exploit this therapeutic vulnerability, we optimized the preclinical use of paxalisib, identifying both precision medicine combination strategies and systemic interventions that reduced side effects and elicited profound tumor control in vivo. Mimicking the patient experience, paxalisib elevated blood glucose levels, promoting the phosphorylation of the insulin receptor (p-INSR) in patient derived DIPG xenograft models. Combining paxalisib with the anti-glycemic drug metformin, synergistically extended the survival of two DIPG xenograft models, decreasing p-Akt, p-mTOR and p-INSR in vivo. Phosphoproteomic profiling of DIPG cells identified paxalisib induced intracellular calcium-regulated PKC signaling. The brain penetrant PKC inhibitor enzastaurin extended the survival of DIPG xenograft models alone, and synergistically combined with paxalisib, enhanced using metformin in three DIPG xenograft models, as well as the immunocompetent H3f3aK27M, PdgfraD842Vmut, Trp53-/- murine brainstem glioma model. Mechanisms of adaptation/plasticity were assessed by ATAC-Seq and quantitative proteomic profiling xenograft tumors refractory to treatment. Here we address the intrinsic neoplastic sequela of DIPG, by combined targeting of PI3K/Akt/mTOR using paxalisib, compensatory PKC signaling using enzastaurin, coupled with strategies to manage treatment-related side-effects and reduced efficacy using metformin; providing the preclinical rationale for the addition of metformin to NCT05009992.

Abstract 5801: Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression

Abstract Primary gliomas arising within the brain are the deadliest form of brain cancer and account for 78% of all malignant brain tumors. Glioma patients have a 5 percent five-year survival rate and drug-resistant tumors often recur following surgical resection and treatment with radiation and/or chemotherapy. The cancer stem cell hypothesis suggests the presence of a subset of undifferentiated cells, namely glioma stem cells (GSCs), in the heterogenous tumor mass responsible for disease progression. Understanding mechanisms responsible for maintaining GSCs and developing strategies to deplete the GSC population could improve glioma treatment and prognosis. Some GSCs are similar to oligodendrocyte precursor cells (OPCs), which are observed in neural development and the adult brain. OPCs are prone to malignant transformation and are believed to be a cell of origin for glioma. Recent findings indicate that the well-characterized neurotransmitter acetylcholine (ACh) maintains the primitive state of normal OPCs via muscarinic ACh receptors (mAChRs) preventing maturation and cell cycle exit. We hypothesized that cholinergic signaling may also maintain the primitive state of OPC-like GSCs. We analyzed publicly available single nuclei RNASeq data of patient glioblastoma samples and observed high expression of CHRM3, encoding the M3 mAChR, in OPC-like cells of the proneural subtype of glioma. Studies in mouse OPC-like GSCs confirmed high levels of CHRM3 expression and demonstrated that ACh generated voltage changes and rapid (< 1 second) increases in cytosolic calcium from internal calcium stores. Exposure to a brain permeant FDA-approved anti-muscarinic drug targeting M3mAChR suppressed proliferation, evoked calcium release, and the activation of intracellular second messengers PKC and ERK. Pharmacologic inhibition of mAChRs in established patient derived glioma grafts prevented re-initiation of tumors in subsequent host animals compared with vehicle treated tumors. These studies suggest that the cholinergic microenvironment maintains GSCs in a manner similar to normal OPCs and provides a platform for repositioning available small molecule mAChR antagonists for treatment of glioma. Citation Format: Sumyuktha V. Anand, Min K. Lee, Alexander G. Skorput, Isabella B. Fox, Alison L. Young, Brock C. Christensen, Allan Gulledge, Matthew C. Havrda. Inhibition of muscarinic acetylcholine receptors in glioma stem cells blocks tumor progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5801.

Larixol inhibits fMLP-induced superoxide anion production and chemotaxis by targeting the βγ subunit of Gi-protein of fMLP receptor in human neutrophils

The over-activated neutrophils through G-protein-coupled receptors (GPCRs) caused inflammation or tissue damage. Therefore, GPCRs or their downstream molecules are major targets for inhibiting uncontrolled neutrophil activation. Our studies investigate the action and underlying mechanism of larixol, a diterpene extract from the root of euphorbia formosana, on fMLP-induced neutrophil respiratory burst, chemotaxis, and granular release. The immunoprecipitation assay was performed to investigate whether larixol inhibits fMLP-induced respiratory burst by interfering with the interaction of fMLP receptor Gi-protein βγ subunits with its downstream molecules. Briefly, larixol inhibited fMLP (0.1 μM)-induced superoxide anion production (IC50:1.98 ± 0.14 μM), the release of cathepsin G (IC50:2.76 ± 0.15 μM) and chemotaxis in a concentration-dependent manner; however, larixol did not inhibit these functions induced by PMA (100 nM). Larixol inhibited fMLP-induced Src kinase phosphorylation. Therefore, larixol attenuated the downstream signaling of Src kinases, ERK1/2, p38, and AKT phosphorylation. Moreover, larixol inhibited fMLP-induced intracellular calcium mobilization, PKC phosphorylation, and p47phox translocation from the cytosol to the plasma membrane. Larixol inhibited the interaction of the βγ subunits of Gi-protein of fMLP receptor with Src kinase or with PLCβ by the immunoprecipitation and duolink assay. Furthermore, larixol did not antagonize the formyl peptide receptors. Larixol did not increase cyclic nucleotide levels in neutrophils. These results suggest that larixol modulated fMLP-induced neutrophils superoxide anion production, chemotaxis, and granular releases by interrupting the interaction of the βγ subunits of Gi-protein with downstream signaling of the fMLP receptor.

CRF-CRFR1 system within the dorsal medial prefrontal cortex are involved in consolation deficits under acute restraint stress in mandarin voles

Consolation is a complex empathic behavior that has recently been observed in some socially living rodents. Despite the growing body of literature suggesting that stress affects some simple form of empathy, the relationship between stress and consolation remains largely understudied. Using monogamous mandarin voles, we found that an acute restraint stress exposure significantly reduced consolation-like behaviors and induced anxiety-like behaviors. Along with these behavioral changes, corticotropin-releasing factor (CRF) and CRF receptor 1 (CRFR1) neurons were activated within the anterior cingulate cortex (ACC) and prelimbic cortex (PrL) but not within the infralimbic cortex (IL). Chemogenetic activation of CRF neurons in the ACC and PrL, recaptured acute stress-induced behavioral dysfunctions. We further observed that intracellular PKA and PKC signaling pathways mediate CRF-induced behavioral dysfunctions, but they work in a regional-specific, sex-biased manner. Together, these results suggest that the local CRF-CRFR1 system within the ACC and PrL is involved in the consolation deficits and anxiety induced by acute stress.

Prenatal hypoxia attenuated contraction of offspring coronary artery associated with decreased PKCβ Ser660 phosphorylation and intracellular calcium

AimsPrenatal hypoxia (PH) could affect peripheral vascular tone of the offspring, thus increasing the risk of cardiovascular diseases in adult. However, it's still unknown whether functions of coronary arteries (COA) in adult offspring would be influenced by PH. The present study aimed at effects of PH on vascular tone of COA and its related mechanisms. MethodsCoronary arteries of adult offspring exposed to hypoxic or normoxic circumstances during gestational day 5 to 21 were collected. Wire myograph system, whole-cell patch clamp technique, IonOptix MyoCam system, PCR, and western blot were used to detect vascular function of adult offspring COA. Key findingsPH significantly attenuated serotonin- and phorbol 12, 13-dibutyrate (PDBu)-induced constriction. Iberiotoxin potentiated PDBu-induced constriction and the effect was augmented by PH, however, no significant differences were found in whole-cell BKCa channel currents and its protein expression. Nifedipine inhibited PDBu-mediated constriction and the inhibitory effect was reduced in PH group, and whole-cell calcium channel current was decreased in offspring COA. Besides, PH reduced the capability of calcium release from the endoplasmic reticulum in COA. The phosphorylated PKCβ protein expression at Ser660 site, not Thr641 site, was significantly decreased in PH offspring. Chronic hypoxia during pregnancy attenuated PDBu-mediated constriction in offspring COA, presumably through decreased phosphorylated PKCβ at serine660 sites and decreased intracellular calcium-related weaker PKC activation. SignificanceThe findings provided new information on the influence of prenatal hypoxia on COA, and suggested potential use of PKCβ-serine660 for early prevention of coronary heart diseases in developmental origins.

Enhancement of acid-sensing ion channel activity by prostaglandin E2 in rat dorsal root ganglion neurons

Prostaglandin E2 (PGE2) and proton are typical inflammatory mediators. They play a major role in pain processing and hypersensitivity through activating their cognate receptors expressed in terminals of nociceptive sensory neurons. However, it remains unclear whether there is an interaction between PGE2 receptors and proton-activated acid-sensing ion channels (ASICs). Herein, we show that PGE2 enhanced the functional activity of ASICs in rat dorsal root ganglion (DRG) neurons through EP1 and EP4 receptors. In the present study, PGE2 concentration-dependently increased ASIC currents in DRG neurons. It shifted the proton concentration-response curve upwards, without change in the apparent affinity of proton for ASICs. Moreover, PGE2 enhancement of ASIC currents was partially blocked by EP1 or EP4 receptor antagonist. PGE2 failed to enhance ASIC currents when simultaneous blockade of both EP1 and EP4 receptors. PGE2 enhancement was partially suppressed after inhibition of intracellular PKC or PKA signaling, and completely disappeared after concurrent blockade of both PKC and PKA signaling. PGE2 increased significantly the expression levels of p-PKCε and p-PKA in DRG cells. PGE2 also enhanced proton-evoked action potentials in rat DRG neurons. Finally, peripherally administration of PGE2 dose-dependently exacerbated acid-induced nocifensive behaviors in rats through EP1 and EP4 receptors. Our results indicate that PGE2 enhanced the electrophysiological activity of ASICs in DRG neurons and contributed to acidosis-evoked pain, which revealed a novel peripheral mechanism underlying PGE2 involvement in hyperalgesia by sensitizing ASICs in primary sensory neurons.

Effects of Paracrine and Intracellular Angiotensin II on Protein Kinase C, MAP kinase ERK1/2, and Nuclear Factor‐κB Signaling Responses in Mouse Glomerular Mesangial cells

Angiotensin II (ANG II) acts not only as a vasoconstrictor, but also as a growth‐promoting cytokine and chemokine, proinflammatory and profibrotic factor in the kidney. Glomerular mesangial cells (MC) are a key target for endocrine, paracrine, autocrine, and intracrine ANG II. Augmentation of local paracrine and intracrine ANG II generation in MCs may induce glomerular injury by promoting MC cell growth, hypertrophy and fibrosis. In the present study, we performed phosphoproteomic characterization of ANG II‐induced protein kinase C (p‐PKCα), MAP kinase ERK1/2 (p‐ERK1/2), p38 MAPK, and nuclear factor‐κB (p65 and p50 subunits) signaling responses in murine glomerular MCs. Murine mesangial cells (MES‐13, ATCC) were grown to ~80% confluence in 6‐well plates in DMEM and Ham's F‐12 medium, containing 5% fetal bovine serum, and 1% penicillin‐streptomycin, maintained at 37°C and 95% O2/5% CO2. After starved at a serum‐free medium for 24 h, MCs were then treated with low to high levels of ANG II (0.1 to 100 nM) for 5 to 20 min, or transfected with a mitochondria‐targeting intracellular ANG II for 48 h (mito‐ECFP/ANG II, 4 μg/well), with or without pretreatment with the AT1 receptor blocker losartan (1 μM), AT2 receptor blocker PD123319 (1 μM) or both blockers. Proteins were extracted for Western blot analysis of p‐PKCα, p‐ERK1/2, p38 MAPK, and p65 and p50 subunits of NF‐κB, respectively. ANG II significantly increased p‐ERK1/2 and p‐PKCα in both dose‐ and time‐dependent manners, with peak responses to ANG II occurred at 0.1 and 1.0 nM, 5–10 min (p<0.05), respectively. ANG II also increased p38 MAPK response, but it peaked at 1 nM, 20 min (p<0.05). By contrast, ANG II appeared to decrease the NF‐κB responses at 1 nM, 10 min, which were reversed by losartan and PD123319. The expression of mito‐ECFP/ANG II selectively in the MC mitochondria significantly increased mitochondrial complex proteins (CI‐CV), which were blocked by losartan (p<0.01). Our results suggest that both paracrine and intracellular ANG II may activate AT1 receptors to induce important intracellular PKC, MAP kinase ERK1/2, p38 MAPK, and NF‐κB signaling responses in MCs, which may contribute to ANG II‐induced glomerular injury in hypertension and kidney diseases.Support or Funding InformationSupported by National Institute of Health grants, 2R01DK067299‐10A1 and 2R01DK102924‐03A1 to Zhuo, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES to Leite.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Trace Amines and the Trace Amine-Associated Receptor 1: Pharmacology, Neurochemistry, and Clinical Implications.

Biogenic amines are a collection of endogenous molecules that play pivotal roles as neurotransmitters and hormones. In addition to the “classical” biogenic amines resulting from decarboxylation of aromatic acids, including dopamine (DA), norepinephrine, epinephrine, serotonin (5-HT), and histamine, other biogenic amines, present at much lower concentrations in the central nervous system (CNS), and hence referred to as “trace” amines (TAs), are now recognized to play significant neurophysiological and behavioral functions. At the turn of the century, the discovery of the trace amine-associated receptor 1 (TAAR1), a phylogenetically conserved G protein-coupled receptor that is responsive to both TAs, such as β-phenylethylamine, octopamine, and tyramine, and structurally-related amphetamines, unveiled mechanisms of action for TAs other than interference with aminergic pathways, laying the foundations for deciphering the functional significance of TAs and its mammalian CNS receptor, TAAR1. Although, its molecular interactions and downstream targets have not been fully elucidated, TAAR1 activation triggers accumulation of intracellular cAMP, modulates PKA and PKC signaling and interferes with the β-arrestin2-dependent pathway via G protein-independent mechanisms. TAAR1 is uniquely positioned to exert direct control over DA and 5-HT neuronal firing and release, which has profound implications for understanding the pathophysiology of, and therefore designing more efficacious therapeutic interventions for, a range of neuropsychiatric disorders that involve aminergic dysregulation, including Parkinson's disease, schizophrenia, mood disorders, and addiction. Indeed, the recent development of novel pharmacological tools targeting TAAR1 has uncovered the remarkable potential of TAAR1-based medications as new generation pharmacotherapies in neuropsychiatry. This review summarizes recent developments in the study of TAs and TAAR1, their intricate neurochemistry and pharmacology, and their relevance for neurodegenerative and neuropsychiatric disease.

Abstract B25: BET bromodomain inhibition targets adaptive responses to lapatinib in HER2-positive breast cancer

Abstract Small molecule kinase inhibitors that target oncogene-driven cancers often elicit dramatic initial clinical responses, but adaptive responses from the kinome and transcriptome limit their efficacy and generate resistance. Such adaptive bypass responses are driven by the disruption of regulatory feedback and feedforward loops that govern many signaling networks. To understand these responses in the context of HER2-addicted cancer, we used a chemical proteomics method to assay whole-kinome activation dynamics in cells treated with the small molecule HER2/EGFR tyrosine kinase inhibitor lapatinib. We observed a rapid reactivation of oncogenic signaling and a global re-wiring of kinase signaling networks including activation of receptor tyrosine kinases (RTKs) HER3, DDR1, FGFRs, IGF1R, INSR, and multiple Ephrin receptors, as well as intracellular nodes PKC, SRC family kinases and FAK. Targeting compensatory kinases with a series of kinase inhibitors provided variable and incremental enhancement of growth inhibition but could not prevent resistant colony formation. Cell line heterogeneity and functional redundancy among compensatory kinases added further difficulty to effectively predict the best combination therapy. This presents a dilemma where combinations of two or even three kinase inhibitors would be insufficient to suppress all the bypass tracks that were rapidly induced by HER2 inhibition. RNAseq revealed a 2-fold dysregulation of approximately 20% of expressed genes within 2 days of lapatinib treatment, with many responsive kinases being transcriptionally upregulated. We argued that in order for lapatinib to have a durable effect, the adaptive response itself must be inhibited. We found that by targeting the BET bromodomain family of chromatin readers, we could suppress the transcriptional upregulation of RTKs that reactivate MAPK/AKT and drive the bypass survival tracks through SRC family kinase and FAK signaling. Treatment of HER2-positive cells with three different small molecule BET bromodomain inhibitors (JQ1, I-BET151, and I-BE762) had little effect on HER2 and downstream signaling pathways in the absence of lapatinib and accordingly allowed resistant colony formation in long-term growth assays. Combining BET bromodomain inhibitors with lapatinib caused a loss of signature RTK expression, prevented MAPK/AKT reactivation, and suppressed resistant colonies from forming. BET bromodomain inhibitors also downregulated responsive RTKs in cells made resistant to lapatinib and thus re-sensitized the cells to lapatinib. Transcriptional profiling defined the effects of BET bromodomain inhibition to be less pronounced than lapatinib, with only 8% of expressed genes being downregulated 2-fold or greater. However, more than 25% of lapatinib-induced genes were downregulated 2-fold, indicating BET bromodomains preferentially modulated the induced gene expression. Current literature describes a crucial role for the BET bromodomain protein BRD4 in the regulation and stability of large enhancer regions (super-enhancers) responsible for oncogene regulation and defining cell fate. ChIPseq analysis of enhancer dynamics in response to lapatinib and JQ1 identified a reorganization of the chromatin landscape within 24 hours of drug treatment, with multiple super-enhancers formed and decommissioned. Importantly, the combination of lapatinib and JQ1 caused a loss of the majority of super-enhancers observed in untreated cells. Together, these results demonstrate epigenetic targeting of adaptive response mechanisms can prevent resistance to small molecule kinase inhibitors and provide a more durable effect for oncogene targeting in the treatment of cancer. Citation Format: Timothy J. Stuhlmiller, Samantha M. Miller, Jon S. Zawistowski, James S. Duncan, Steven P. Angus, Deborah A. Granger, Rachel A. Reuther, Kyla A.L. Collins, Gomez M. Shawn, Pei-Fen Kuan, Xin Chen, Noah Sciaky, Gary L. Johnson. BET bromodomain inhibition targets adaptive responses to lapatinib in HER2-positive breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B25.

CTRP3 Stimulates Proliferation and Anti-Apoptosis of Prostate Cells through PKC Signaling Pathways.

C1q/TNF-related protein-3 (CTRP3) is a novel adipokine with roles in multiple cellular processes. However, little is known about its function in prostate cells. This study investigated the effects and mechanisms of CTRP3 in prostate cells. We first generated and purified CTRP3 protein in HEK 293T cells. Proliferation of RWPE-1 prostate cells was evaluated by MTT analyses under treatment with different concentrations of CTRP3 for various exposure times. The results revealed maximum enhancement of proliferation with 10 μg/mL CTRP3 for 72 h. Cell apoptosis and cell cycle were determined by TUNEL staining and flow cytometry analysis. TUNEL assay showed decreased TUNEL-positive cells in RWPE-1 prostate cells treated with CTRP3, and flow cytometry showed significantly decreased apoptotic cells upon CTRP3 treatment (treated cells, 8.34±1.175 vs. controls, 20.163±0.35) (P < 0.01). Moreover, flow cytometry analysis also showed a significant decrease of cells in the G1 phase and an increase of cells in the S and G2 phase upon CTRP3 treatment (treated cells, 42.85±1.40 vs. control, 52.77±0.90; 28.41±0.57 vs. 23.49±1.13; 27.08±1.97 vs. 22.20±1.32, respectively) (all P < 0.05). Two-dimensional gel electrophoresis and mass spectrometry identified differentially expressed proteins, including cytokeratin-19, GLRX3 and DDAH1, which were upregulated in CTRP3 treated cells, and cytokeratin-17 and 14-3-3 sigma, which were downregulated. GLRX3, DDAH1 and 14-3-3 sigma were confirmed using western blot analysis. A PKC inhibitor, staurosporine, was used to inhibit PKC activity in CTRP3 treated RWPE-1 cells. Staurosporine completely abolished the CTRP3-induced increased phosphorylation of intracellular PKC substrates and CTRP3-stimulated effect by RWPE-1 cells. Our results provide the first evidence for a physiological role of the novel adipokine, CTRP3, in prostate cells. Our findings suggest that CTRP3 could improve proliferation and anti-apoptosis of prostate cells through protein kinase C signaling pathways.

Potentiation of tonic GABAergic inhibition by activation of postsynaptic kainate receptors

Presynaptic kainate-type glutamate ionotropic receptors (KARs) that mediate either the depression or the facilitation of GABA release have been intensively studied. Little attention has been given to the modulation of GABAA receptors (GABAARs) by postsynaptic KARs. Recent studies suggest that two GABAAR populations, synaptic (sGABAAR) and extrasynaptic (eGABAAR) GABAARs, mediate phasic and tonic forms of inhibition, respectively. Tonic inhibition plays an important role in the excitability of neuronal circuits and the occurrence of epileptic seizures. For this study, we are the first to report that the activation of postsynaptic KARs by the KAR agonist, Kainic acid (KA, 5μM), enhanced tonic inhibition by potentiating eGABAARs. KA enhanced THIP-induced eGABAAR currents and prolonged the rise and decay time of muscimol-induced sGABAAR/eGABAAR currents, but also depressed the amplitude of evoked inhibitory postsynaptic currents (IPSCs), unitary IPSCs (uIPSCs), and muscimol-induced sGABAAR/eGABAAR currents. The PKC inhibitor, staurosporine (1μM), in the patch pipette solution fully blocked the KA-induced potentiation of tonic inhibition, suggesting the involvement of an intracellular PKC pathway. Our study suggests that the activation of postsynaptic KARs potentiates eGABAARs but depresses sGABAARs. By activating postsynaptic KARs, synaptically released glutamate depresses phasic inhibition to facilitate neuronal plasticity, but potentiates tonic inhibition to protect neurons from over-excitation.

Acute effect of 3β-hidroxihop-22(29)ene on insulin secretion is mediated by GLP-1, potassium and calcium channels for the glucose homeostasis

The effect of 3β-hidroxihop-22(29)ene (3-BHO) on insulin and glucagon-like peptide 1 (GLP-1) secretion as well as the mechanism of action of the compound in pancreatic islet on glucose homeostasis was investigated. The data from in vivo treatment show that 3-BHO significantly reduces the hyperglycemia by increasing the insulin and GLP-1 secretion, as well as by accumulating hepatic glycogen in hyperglycemic rats. In rat pancreatic β-cell, 3-BHO stimulates the glucose uptake, insulin vesicles translocation to the plasma membrane and thus the insulin secretion through the involvement of potassium channels (ATP- and Ca2+-dependent K+ channels) and calcium channels (L-type voltage-dependent calcium channels (L-VDCC)). Furthermore, this study also provides evidence for a crosstalk between intracellular high calcium concentration, PKA and PKC in the signal transduction of 3-BHO to stimulate insulin secretion. In conclusion, 3-BHO diminishes glycaemia, stimulates GLP-1 secretion and potentiates insulin secretion and increase hepatic glycogen content. Moreover, this triterpene modulates calcium influx characterizing ATP-K+, Ca2+–K+ and L-VDCC channels-dependent pathways as well as PKA and PKC activity in pancreatic islets underlying the signaling of 3-BHO for the secretory activity and contribution on glucose homeostasis.

Pyk2 contributes to reepithelialization by promoting MMP expression. Focus on “Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice”

matrix metalloproteinases (MMPs) play an important role in wound healing (reviewed in Ref. [5][1]). In the skin, the hemidesmosome attachment between keratinocytes and basement membrane is disrupted by MMP activity, so that keratinocytes are released to migrate under the wound matrix and contact the

Real-time monitoring of intracellular signal transduction in PC12 cells by two-dimensional surface plasmon resonance imager

Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. The conventional methods used to observe intracellular reactions have not been convenient with several steps such as labeling and washing steps prior to the readout. Consequently, there is a critical need for label-free observation techniques for monitoring intracellular reactions. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via local refractive index change in PC12 cells adhered on a gold sensor slide without any indicator reagent. PC12 cells were stimulated with KCl and phorbol-12-myristate-13-acetate (PMA, a protein kinase C [PKC] activator) at different concentrations in order to induce intracellular PKC translocation. 2D–SPR signal (reflection intensity change) is very consistent with the cellular response normally detected for these stimulants. Our results suggest that complex intracellular reactions could be real-time monitored and characterized by the 2D–SPR imager. It is further expected that signal transmission that was followed by the translocation of signaling proteins could be observed at the single cell level with the high-resolution 2D–SPR imager.

Glycated collagen and altered glucose increase endothelial cell adhesion strength

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen-coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm(2) in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength αv β3 integrins to bind to glycated collagen instead of the typical α2 β1 integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm(2) higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis.

Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells by the Traceable Kinase Method

A previous report [Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370] described the application of the traceable kinase method in identifying substrates of protein kinase Cα (PKC-α) in nontransformed human breast MCF-10A cells. Here, a nonradioactive variation of this method compared the phosphoprotein profiles of three traceable PKC isoforms (α, δ, and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high-affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N(6)-phenyl-ATP, and the resulting phosphoproteins were analyzed by Western blotting with an antibody that recognizes the phosphorylated PKC consensus site. Phosphoprotein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, -δ, and -ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (moles of phospho-CEP4 per mole of CEP4). Following knockdown with isoform-specific shRNA-encoding plasmids, the level of phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC-α, -δ, and -ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and -δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms.

The role of sex steroids on cellular events involved in vascular disease

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.

Calcium regulation of keratinocyte differentiation

Calcium is the major regulator of keratinocyte differentiation in vivo and in vitro. A calcium gradient within the epidermis promotes the sequential differentiation of keratinocytes as they traverse the different layers of the epidermis to form the permeability barrier of the stratum corneum. Calcium promotes differentiation by both outside–in and inside–out signaling. A number of signaling pathways involved with differentiation are regulated by calcium, including the formation of desmosomes, adherens junctions and tight junctions, which maintain cell–cell adhesion and play an important intracellular signaling role through their activation of various kinases and phospholipases that produce second messengers that regulate intracellular free calcium and PKC activity, critical for the differentiation process. The calcium receptor plays a central role by initiating the intracellular signaling events that drive differentiation in response to extracellular calcium. This review will discuss these mechanisms.