Phosphoinositide Signaling Pathway Research Articles

Multimodal action of KRP203 on phosphoinositide kinases in vitro and in cells

Increased phosphoinositide signaling is commonly associated with cancers. While “one-drug one-target” has been a major drug discovery strategy for cancer therapy, a “one-drug multi-targets” approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIβ) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kβ, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3 h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3,5)P2, and PI(3,4,5)P3 levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways.

Regulation of B-1 cell numbers and B cell-mediated antibody production by Inpp4b.

T and B lymphocytes are crucial players in cellular and humoral immune responses. The development, activation and differentiation of T and B lymphocytes are regulated by the best characterized PI3K-PI (3,4,5) P3-AKT phosphoinositide signalling pathway. As a branch of the phosphoinositide signalling pathway, the lipid phosphatase INPP4B inhibits AKT activation through degrading the phosphoinositide signalling messenger PI (3,4) P2. However, the role of Inpp4b in T and B lymphocytes remains elusive. Here, we reported that Inpp4b was highly expressed in human and murine T- and B-1 lymphocytes. Despite its higher expression in T lymphocytes, neither T cell development and homeostasis nor in vitro T cell activation and CD4+ T cell differentiation were altered upon loss of Inpp4b. Interestingly, combined direct phenotype analysis of Inpp4b conventional knockout mice and adoptive transfer studies revealed that ablation of Inpp4b intrinsically reduced peritoneal B-1 cells rather B-2 cells. Moreover, Inpp4b deficiency led to impaired thymus independent (TI) and thymus dependent (TD) antigens-induced antibody production. Further in vitro analysis revealed that CD40-mediated B cell proliferation was impaired upon ablation of Inpp4b. Our findings reveal that Inpp4b is required in regulating B-1 cell numbers and B cell-mediated antibody production.

Immunocytochemistry of phospholipase D1 and D2 in cultured cells

Activation of Gq protein-coupled receptors triggers the phospholipase C (PLC) pathway, which yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA), which serves as another second messenger. Along with PLC-DGK pathway, PA is produced directly by the action of phospholipase D (PLD), which hydrolyzes the major membrane phospholipid: phosphatidylcholine (PC). PA is converted to DG by phosphatidic acid phosphatase, suggesting that PLD, together with DGK, is a key enzyme regulating DG and PA. PLD has been implicated in a broad range of cellular processes. However, cellular expression and subcellular localization of PLD remain elusive because of a lack of specific antibodies against PLDs. For this study, we raised specific antibodies against major mammalian PLD isoforms: PLD1 and PLD2. Immunocytochemical analysis using specific antibodies showed clearly that native PLD1 and PLD2 localize to distinct subcellular regions as dot-like structures in cultured cells. PLD1 predominantly localizes to the plasma membrane, whereas PLD2 mostly localizes within the cytoplasm. These findings suggest that PLD1 and PLD2 have different roles in the phosphoinositide signaling pathway in distinct subcellular regions.

Expression GWAS of PGIP1 Identifies STOP1-Dependent and STOP1-Independent Regulation of PGIP1 in Aluminum Stress Signaling in Arabidopsis.

To elucidate the unknown regulatory mechanisms involved in aluminum (Al)-induced expression of POLYGALACTURONASE-INHIBITING PROTEIN 1 (PGIP1), which is one of the downstream genes of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) regulating Al-tolerance genes, we conducted a genome-wide association analysis of gene expression levels (eGWAS) of PGIP1 in the shoots under Al stress using 83 Arabidopsis thaliana accessions. The eGWAS, conducted through a mixed linear model, revealed 17 suggestive SNPs across the genome having the association with the expression level variation in PGIP1. The GWAS-detected SNPs were directly located inside transcription factors and other genes involved in stress signaling, which were expressed in response to Al. These candidate genes carried different expression level and amino acid polymorphisms. Among them, three genes encoding NAC domain-containing protein 27 (NAC027), TRX superfamily protein, and R-R-type MYB protein were associated with the suppression of PGIP1 expression in their mutants, and accordingly, the system affected Al tolerance. We also found the involvement of Al-induced endogenous nitric oxide (NO) signaling, which induces NAC027 and R-R-type MYB genes to regulate PGIP1 expression. In this study, we provide genetic evidence that STOP1-independent NO signaling pathway and STOP1-dependent regulation in phosphoinositide (PI) signaling pathway are involved in the regulation of PGIP1 expression under Al stress.

The Mechanisms Underlying the Protective Action of Selenium Nanoparticles against Ischemia/Reoxygenation Are Mediated by the Activation of the Ca2+ Signaling System of Astrocytes and Reactive Astrogliosis.

In recent years, much attention has been paid to the study of the therapeutic effect of the microelement selenium, its compounds, especially selenium nanoparticles, with a large number of works devoted to their anticancer effects. Studies proving the neuroprotective properties of selenium nanoparticles in various neurodegenerative diseases began to appear only in the last 5 years. Nevertheless, the mechanisms of the neuroprotective action of selenium nanoparticles under conditions of ischemia and reoxygenation remain unexplored, especially for intracellular Ca2+ signaling and neuroglial interactions. This work is devoted to the study of the cytoprotective mechanisms of selenium nanoparticles in the neuroglial networks of the cerebral cortex under conditions of ischemia/reoxygenation. It was shown for the first time that selenium nanoparticles dose-dependently induce the generation of Ca2+ signals selectively in astrocytes obtained from different parts of the brain. The generation of these Ca2+ signals by astrocytes occurs through the release of Ca2+ ions from the endoplasmic reticulum through the IP3 receptor upon activation of the phosphoinositide signaling pathway. An increase in the concentration of cytosolic Ca2+ in astrocytes leads to the opening of connexin Cx43 hemichannels and the release of ATP and lactate into the extracellular medium, which trigger paracrine activation of the astrocytic network through purinergic receptors. Incubation of cerebral cortex cells with selenium nanoparticles suppresses ischemia-induced increase in cytosolic Ca2+ and necrotic cell death. Activation of A2 reactive astrocytes exclusively after ischemia/reoxygenation, a decrease in the expression level of a number of proapoptotic and proinflammatory genes, an increase in lactate release by astrocytes, and suppression of the hyperexcitation of neuronal networks formed the basis of the cytoprotective effect of selenium nanoparticles in our studies.

Activation of Cx43 Hemichannels Induces the Generation of Ca2+ Oscillations in White Adipocytes and Stimulates Lipolysis.

The aim of the study was to investigate the mechanisms of Ca2+ oscillation generation upon activation of connexin-43 and regulation of the lipolysis/lipogenesis balance in white adipocytes through vesicular ATP release. With fluorescence microscopy it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+ responses in white adipocytes: Ca2+ oscillations and transient Ca2+ signals. It was found that activation of the connexin half-channels is involved in the generation of Ca2+ oscillations, since the blockers of the connexin hemichannels—carbenoxolone, octanol, proadifen and Gap26—as well as Cx43 gene knockdown led to complete suppression of these signals. The activation of Cx43 in response to the reduction of [Ca2+]ex was confirmed by TIRF microscopy. It was shown that in response to the activation of Cx43, ATP-containing vesicles were released from the adipocytes. This process was suppressed by knockdown of the Cx43 gene and by bafilomycin A1, an inhibitor of vacuolar ATPase. At the level of intracellular signaling, the generation of Ca2+ oscillations in white adipocytes in response to a decrease in [Ca2+]ex occurred due to the mobilization of the Ca2+ ions from the thapsigargin-sensitive Ca2+ pool of IP3R as a result of activation of the purinergic P2Y1 receptors and phosphoinositide signaling pathway. After activation of Cx43 and generation of the Ca2+ oscillations, changes in the expression levels of key genes and their encoding proteins involved in the regulation of lipolysis were observed in white adipocytes. This effect was accompanied by a decrease in the number of adipocytes containing lipid droplets, while inhibition or knockdown of Cx43 led to inhibition of lipolysis and accumulation of lipid droplets. In this study, we investigated the mechanism of Ca2+ oscillation generation in white adipocytes in response to a decrease in the concentration of Ca2+ ions in the external environment and established an interplay between periodic Ca2+ modes and the regulation of the lipolysis/lipogenesis balance.

PIP5K2A (rs10828317) polymorphism in patients with alcohol dependence and affective disorders

Alcohol dependence (AD) and affective disorders (ADs) are serious medical and socio-economic problems of modern society. It is hypothesized that both disorders share a common neurobiological basis. Phosphatidylinositol-4-phosphate-5-kinase type 2 alpha (PIP5K2A) plays an essential role in neuronal phosphoinositide signaling pathways. Nonsynonymous rs10828317 mutation of the PIP5K2A gene leads to conformational changes of the PIP5K2A protein and a decrease in the functional activity of this enzyme. In this study, we assessed the possibility of using the rs10828317 polymorphic variant of the PIP5K2A gene as a marker of AD and ADs.Objective: to study the associations of the PIP5K2A (rs10828317) polymorphism with AD and ADs clinical course.Patients and methods. We enrolled 255 patients with AD and 325 patients with ADs. 126 patients with AD and 71 patients with ADs underwent a comprehensive clinical, clinical-dynamic, psychodiagnostic assessment using a set of clinical scales and tests, including Structured Interview Guide For The Hamilton Depression Rating Scale, Seasonal Affective Disorders Version (SIGH-SAD), Alcohol Use Disorders Identification Test (AUDIT), The Obsessive-Compulsive Drinking Scale for craving in alcohol (OCDS).Results and discussion. PIP5K2A (rs10828317) polymorphism in patients with AD was associated with the OCDS mean score after the inpatient treatment; in patients with ADs - with the severity of atypical depression symptoms assessed by SIGH-SAD at the time of admission.Conclusion. The results of our pilot study indicate the involvement of PIP5K2A (rs10828317) polymorphism in the AD and ADs pathophysiology.

Structural insights into C1-ligand interactions: Filling the gaps by in silico methods.

Protein Kinase C isoenzymes (PKCs) are the key mediators of the phosphoinositide signaling pathway, which involves regulated hydrolysis of phosphatidylinositol (4,5)-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate. Dysregulation of PKCs is implicated in many human diseases making this class of enzymes an important therapeutic target. Specifically, the DAG-sensing cysteine-rich conserved homology-1 (C1) domains of PKCs have emerged as promising targets for pharmaceutical modulation. Despite significant progress, the rational design of the C1 modulators remains challenging due to difficulties associated with structure determination of the C1-ligand complexes. Given the dearth of experimental structural data, computationally derived models have been instrumental in providing atomistic insight into the interactions of the C1 domains with PKC agonists. In this review, we provide an overview of the in silico approaches for seven classes of C1 modulators and outline promising future directions.

Structural Basis of Protein Kinase C Tumor‐Promoting Response And Membrane Recruitment

Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. The hallmark of their activation is translocation to lipid membranes. Dysregulation of PKC signaling has been implicated in cancer progression, cardiac disease, and Alzheimer’s disease. One of the major challenges in the field is to understand the structural basis of PKC activation and regulation ‐‐ at the level of atomic resolution.Here, we applied NMR and FRET spectroscopy to gain insight into the structure and protein‐membrane interactions of the regulatory domain of PKC that consist of three peripheral membrane modules: the tandem conserved homology‐1 (C1) and conserved homology‐2 (C2) domains. The C1 domains undergo membrane insertion upon binding their natural agonist, diacylglycerol, as well as tumor‐promoting phorbol esters and other small‐molecule agonists. C2 associates with anionic membranes upon binding Ca2+. Simultaneous association of all domains with membranes ensures the coincidence of four signals: Ca2+ ions, diacylglycerol, phosphatidylserine, and phosphatidylinositol‐4,5‐bisphosphate.Here, we report the first structure of the C1 domain complexed to a potent tumor‐promoting agent, phorbol 12,13‐dibutyrate (PDBu), in the membrane‐mimicking environment. The structure of the complex shows that C1 has to undergo a conformational change in order to capture the membrane‐embedded ligand. The structure also revealed the role of individual amino acids in the interactions with PDBu and membrane‐mimicking environment. To probe how different agonists modulate the interaction of C1 with membranes, we conducted NMR paramagnetic relaxation enhancement (PRE) experiments with bicelles doped with paramagnetic lipid. The PRE patterns revealed that the C1 domains adopt tilted rather than vertical orientation in the membrane. Our data on four different agonists indicate that C1 domains dictate the general orientation of the agonist complexes in the membrane, but the agonists determine the depth of insertion. This provides a plausible explanation for the differences in the potency of these ligands to achieve the acute and chronic activation response of PKC.To gain insight into the mechanism of coincidence detection, we developed protein‐to‐membrane FRET assay that we used to characterize the interplay of the C1 and C2 domains in the membrane association step. The FRET experiments revealed a clear picture of how tumor‐promoting agents can elicit constitutive activation of PKC through simultaneous recruitment of C1 domains to membranes and sensitization of the C2 domain to Ca2+.Support or Funding InformationNIH R01 GM108998; NSF CHE‐1905116; Welch Foundation A‐1784Agonist‐guided membrane interaction of C1B domain of PKCδ studied by paramagnetic NMR. (A) Schematic representation of the approach. (B) Chemical representations of the different agonists used in the study.Figure 1

Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus Replication and Phosphatidylinositol 4-Phosphate Enrichment at the Viral Replication Organelle.

The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication.IMPORTANCE Hepatitis C virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate [PI(4)P] in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol-binding protein (OSBP). OSBP exchanges PI(4)P with cholesterol, thus removing PI(4)P from the HCV RO. Here, we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP and may replenish PI at the HCV RO by interacting with VAMP-associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, and VAPs completes our understanding of the phosphoinositide cycle between the ER and HCV ROs.

Phosphoinositides: Regulators of Nervous System Function in Health and Disease.

Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol have emerged as regulators of key sub-cellular processes such as membrane transport, cytoskeletal function and plasma membrane signaling in eukaryotic cells. All of these processes are also present in the cells that constitute the nervous system of animals and in this setting too, these are likely to tune key aspects of cell biology in relation to the unique structure and function of neurons. Phosphoinositides metabolism and function are mediated by enzymes and proteins that are conserved in evolution, and analysis of knockouts of these in animal models implicate this signaling system in neural function. Most recently, with the advent of human genome analysis, mutations in genes encoding components of the phosphoinositide signaling pathway have been implicated in human diseases although the cell biological basis of disease phenotypes in many cases remains unclear. In this review we evaluate existing evidence for the involvement of phosphoinositide signaling in human nervous system diseases and discuss ways of enhancing our understanding of the role of this pathway in the human nervous system’s function in health and disease.

PIP4K2A and PIP4K2C transcript levels are associated with cytogenetic risk and survival outcomes in acute myeloid leukemia

Phosphoinositide signaling pathway orchestrates primordial molecular and cellular functions in both healthy and pathologic conditions. Phosphatidylinositol-5-phosphate 4-kinase type 2 lipid kinase (PIP4K2) family, which compromises PIP4K2A, PIP4K2B and PIP4K2C, has drawn the attention in human cancers. Particularly in hematological malignancies, PIP4K2A was already described as an essential protein for a malignant phenotype, although the clinical and biological impact of PIP4K2B and PIP4K2C proteins have not being explored in the same extent. In the present study, we investigated the impact on clinical outcomes and gene network of PIP4K2A, PIP4K2B and PIP4K2C mRNA transcripts in acute myeloid leukemia (AML) patients included in The Cancer Genome Atlas (2013) study. Our results indicate that PIP4K2A and PIP4K2C, but not PIP4K2B, mRNA levels were significantly reduced in AML patients assigned to the favorable risk group (p < 0.05) and low levels of PIP4K2A and PIP4K2C positively affect clinical outcomes of AML patients (p < 0.05). Gene set enrichment analyses indicate that the expression of PIP4K2 genes is associated with biological process such as signal transduction, metabolism of RNA and genomic instability related-gene sets. In summary, our study provides additional evidence of the involvement of members of the PIP4K2 family, in particular PIP4K2A and PIP4K2C, in AML.

Role of Myo‐inositol in Ischemic Stroke Outcome in a Preclinical Tobacco Smoke Exposed Mouse Model

Tobacco smoking is a preventable risk factor for ischemic stroke in both males and females and contributes up to 14% of all stroke deaths. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome, such as seen in tobacco smokers. Myo‐inositol (MI) is a major organic “non‐perturbing” osmolyte that promotes cell membrane stability and also a precursor for phosphoinositide signaling pathway. Our previous work reported that MI attenuated brain infarct and edema ratio in an ischemic stroke model coupled to preclinical diabetes and suggests that MI and MI transporters provide neuroprotection during/following stroke both in non‐diabetic and diabetic conditions through protection from brain edema. Since our lab has shown that tobacco smoke exposure worsens stroke edema similarly to diabetic conditions, the aim of the present study was to assess the neuroprotective effects of MI associated with ischemic mice exposed to tobacco smoke. In this study, we also investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of astrocytes cells, which are the most abundant glial cells in the brain and play an important role in post‐stroke reorganization, and indirectly protecting neuronal metabolic support. Primary mouse astrocytes were incubated with tobacco smoke extract (TSE) overnight prior for in vitro studies. Toxicity of TSE was measured using MTT assay after incubating astrocytes with TSE 6hr and 24hr. Therapeutic efficacy of MI was evaluated in a transient‐middle cerebral artery occlusion (tMCAO) mouse model after smoking 3R4F research cigarettes for 10 days. Uptake studies showed an increase of MI uptake increased with longer incubation of TSE in normoxic conditions. Additionally, it was also observed that uptake of MI increased during Oxygen Glucose Deprivation(OGD) conditions with prior exposure of TSE. These results are in support with our previous reports, where we found an increased expression of MI transports, Sodium Myo‐inositol Transporter 1 (SMIT1) and Sodium Dependent Glucose Transporter 6 (SGLT6) during OGD exposure. Preliminary animal studies using middle cerebral artery occlusion mouse model for in vivo stroke studies, demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in tobacco smoke exposed animals compared to non‐tobacco smoked ischemic animals and also improved motor function in behavioral tests. Taken together with previous work, this study provides an insight into how MI may play a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome, especially in tobacco smoke populations. Further studies are required to understand the neuroprotective mechanisms of MI in ischemic stroke injury in reducing brain edema.Support or Funding InformationR01NS076012 and R01DA029121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Amphetamine-induced Conditioned Place Preference and Changes in mGlu1/5 Receptor Expression and Signaling in the Rat Medial Prefrontal Cortex

The medial prefrontal cortex (mPFC) is implicated in the rewarding effect of psychostimulants, although molecular mechanisms underlying the rewarding properties of stimulants in this region are poorly understood. Group I metabotropic glutamate (mGlu) receptors (mGlu1/5 subtypes) are believed to be critical in this event. We thus in this study investigated changes in mGlu1/5 receptor expression and function in the rat mPFC in response to conditioned place preference (CPP) induced by amphetamine. Repeated amphetamine administration (2.5 mg/kg, once daily on alternate days for 10 days) induced reliable CPP. In the mPFC, surface expression of mGlu5 receptors was elevated in rats after amphetamine conditioning. mGlu5 receptors were also increased at synaptic and extrasynaptic sites in amphetamine-conditioned rats. Expression of mGlu1 receptors was stable in surface and synaptic compartments, while it was elevated in the extrasynaptic location. In mPFC neurons, the mGlu1/5 agonist-stimulated phosphoinositide signaling pathway was upregulated in its efficacy following amphetamine conditioning. The mGlu1/5 agonist-stimulated Src kinase phosphorylation was also augmented in rats treated with amphetamine. These results demonstrate the sensitivity of mPFC mGlu1/5 receptors to amphetamine-induced CPP. Amphetamine conditioning results in the upregulation of mGlu1/5 receptor expression at subcellular and/or subsynaptic levels and mGlu1/5-mediated postreceptor signaling in mPFC neurons.

Light-Induced Opening of the TRP Channel in Isolated Membrane Patches Excised from Photosensitive Microvilli from Drosophila Photoreceptors

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry–DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.

RasGRP3, a Ras guanyl releasing protein 3 that contributes to malignant proliferation and aggressiveness in human esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide; however, clinical and pathological parameters have limited ability in discriminating between clinically significant and indolent ESCC. Since RasGRP3 transcript levels have prognostic value in discriminating ESCC with different clinical aggressiveness, we decided to investigate its putative oncogenic role in ESCC. We found that RasGRP3 was highly expressed in ESCC cells. Suppression of endogenous RasGRP3 expression in esophageal cell lines reduced Ras-GTP formation as well as AKT phosphorylation. RasGRP3 suppression also inhibited cell invasion and migration and reduced proliferation, demonstrating the importance of RasGRP3 for the transformed phenotype of melanoma cells. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth and migration, confirming the functional role of RasGRP3 in the altered behaviour of these cells. This suggests that RasGRP3 may function as a Ras activator in the phosphoinositide signalling pathway and may potentially serve as a new therapeutic target.

Relationship Between INPP5E Gene Expression and Embryonic Neural Development in a Mouse Model of Neural Tube Defect.

BackgroundThe INPP5E gene encodes for the inositol polyphosphate-5-phosphatase (INPP5E) 72 kDa protein that regulates the phosphoinositide signaling pathway and other cellular activities, but the functional role of this gene in embryonic neurodevelopment and neural tube defect (NTD) remains unclear. The aim of this study was to use a mouse model of NTD to investigate the expression levels of the INPP5E gene during neural development and the occurrence of NTD.Material/MethodsIn an established NTD mouse model, stereoscopy was used to look for morphological defects. Transcription and expression levels of the INPP5E gene in neural tissues were detected using real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blotting in the NTD mouse embryos and compared with control mouse embryos.ResultsThe expression levels of the INPP5E gene decreased as embryonic development progressed in the neural tissue of control mice embryos, but showed no obvious trend in the neural tissues of the NTD mouse embryos. The expression levels of the INPP5E gene in NTD mouse embryos were significantly lower compared with control embryos, at the time of neural tube closure (gestational day 11.5).ConclusionsThe INPP5E gene regulates the process of embryonic neural development. Abnormal levels of expression of the INPP5E gene may contribute to NTDs. Increased knowledge of the expression pattern of the INPP5E gene may lead to an advanced understanding of the molecular mechanism of embryonic neurodevelopment and identify more specific directions to explore potential treatments for NTDs associated with abnormalities in INPP5E gene expression levels.

Alterations in mGlu5 receptor expression and function in the striatum in a rat depression model.

Major depressive disorder is a common form of mental illness. Many brain regions are implicated in the pathophysiology and symptomatology of depression. Among key brain areas is the striatum that controls reward and mood and is involved in the development of core depression-like behavior in animal models of depression. While molecular mechanisms in this region underlying depression-related behavior are poorly understood, the glutamatergic input to the striatum is believed to play a role. In this study, we investigated changes in metabotropic glutamate (mGlu) receptor expression and signaling in the striatum of adult rats in response to prolonged (10-12weeks) social isolation, a pre-validated animal paradigm modeling depression in adulthood. We found that mGlu5 receptor protein levels in the striatum were increased in rats that showed typical depression- and anxiety-like behavior after chronic social isolation. This increase in mGlu5 receptor expression was seen in both subdivisions of the striatum, the nucleus accumbens and caudate putamen. At subcellular and subsynaptic levels, mGlu5 receptor expression was elevated in surface membranes at synaptic sites. In striatal neurons, the mGlu5-associated phosphoinositide signaling pathway was augmented in its efficacy after prolonged social isolation. These data indicate that the mGlu5 receptor is a sensitive substrate of depression. Adulthood social isolation leads to the up-regulation of mGlu5 receptor expression and function in striatal neurons.

Modulation of Ras/ERK and Phosphoinositide Signaling by Long-Chain n-3 PUFA in Breast Cancer and Their Potential Complementary Role in Combination with Targeted Drugs.

A potential complementary role of the dietary long-chain n-3 polyunsaturated fatty acids (LCn-3 PUFA) in combination with innovative mono-targeted therapies has recently been proposed. These compounds are thought to act pleiotropically to prevent the development and progression of a variety of cancers, including breast cancer. We hereinafter critically analyze the reports investigating the ability of LCn-3 PUFA to modulate the Ras/ERK and the phosphoinositide survival signaling pathways often aberrantly activated in breast cancer and representing the main targets of innovative therapies. The in vitro or in vivo animal and human interventional studies published up to January 2017 investigating the effects of LCn-3 PUFA on these pathways in normal and cancerous breast cells or tissues were identified through a systematic search of literature in the PubMed database. We found that, in most cases, both the in vitro and in vivo studies demonstrated the ability of LCn-3 PUFA to inhibit the activation of these pro-survival pathways. Altogether, the analyzed results strongly suggest a potential role of LCn-3 PUFA as complementary agents in combination with mono-targeted therapies. Moreover, the results indicate the need for further in vitro and human interventional studies designed to unequivocally prove the potential adjuvant role of these fatty acids.

Vitamin D and Depression: Cellular and Regulatory Mechanisms.

Depression is caused by a change in neural activity resulting from an increase in glutamate that drives excitatory neurons and may be responsible for the decline in the activity and number of the GABAergic inhibitory neurons. This imbalance between the excitatory and inhibitory neurons may contribute to the onset of depression. At the cellular level there is an increase in the concentration of intracellular Ca2+ within the inhibitory neurons that is driven by an increase in entry through the NMDA receptors (NMDARs) and through activation of the phosphoinositide signaling pathway that generates inositol trisphosphate (InsP3) that releases Ca2+ from the internal stores. The importance of these two pathways in driving the elevation of Ca2+ is supported by the fact that depression can be alleviated by ketamine that inhibits the NMDARs and scopolamine that inhibits the M1 receptors that drive InsP3/Ca2+ pathway. This increase in Ca2+ not only contributes to depression but it may also explain why individuals with depression have a strong likelihood of developing Alzheimer's disease. The enhanced levels of Ca2+ may stimulate the formation of Aβ to initiate the onset and progression of Alzheimer's disease. Just how vitamin D acts to reduce depression is unclear. The phenotypic stability hypothesis argues that vitamin D acts by reducing the increased neuronal levels of Ca2+ that are driving depression. This action of vitamin D depends on its function to maintain the expression of the Ca2+ pumps and buffers that reduce Ca2+ levels, which may explain how it acts to reduce the onset of depression.