Inositol Phospholipid Research Articles

Inositol lipids: from an archaeal origin to phosphatidylinositol 3,5-bisphosphate faults in human disease

The last couple of decades have seen an extraordinary transformation in our knowledge and understanding of the multifarious biological roles of inositol phospholipids. Herein, I briefly consider two topics. The first is the role that recently acquired biochemical and genomic information-especially from archaeons-has played in illuminating the possible evolutionary origins of the biological employment of inositol in lipids, and some questions that these studies raise about the 'classical' biosynthetic route to phosphatidylinositol. The second is the growing recognition of the importance in eukaryotic cells of phosphatidylinositol 3,5-bisphosphate. Phosphatidylinositol 3,5-bisphosphate only entered our phosphoinositide consciousness quite recently, but it is speedily gathering a plethora of roles in diverse cellular processes and diseases thereof. These include: control of endolysosomal vesicular trafficking and of the activity of ion channels and pumps in the endolysosomal compartment; control of constitutive and stimulated protein traffic to and from plasma membrane subdomains; control of the nutrient and stress-sensing target of rapamycin complex1 pathway (TORC1); and regulation of key genes in some central metabolic pathways.

A Phosphatidylinositol Species Acutely Generated by Activated Macrophages Regulates Innate Immune Responses

Activation of macrophages with stimuli of the innate immune response results in the intense remodeling of arachidonate-containing phospholipids, leading to the mobilization of large quantities of this fatty acid for conversion into biologically active eicosanoids. As a consequence of this process, the arachidonate levels in membrane phospholipids markedly decrease. We have applied mass spectrometry-based lipid profiling to study the levels of arachidonate-containing phospholipids under inflammatory activation of macrophages. We identify an unusual inositol phospholipid molecule, PI(20:4/20:4), the levels of which do not decrease but actually increase by 300% after activation of the macrophages. PI(20:4/20:4) is formed and degraded rapidly, suggesting a role for this molecule in regulating cell signaling events. Using a metabolipidomic approach consisting in exposing the cells to deuterium-labeled arachidonate at the time they are exposed to stimuli, we show that PI(20:4/20:4) biosynthesis occurs via the sequential incorporation of arachidonate, first into the sn-2 position of a preformed phosphatidylinositol (PI) molecule, followed by the rapid introduction of a second arachidonate moiety into the sn-1 position. Generation requires the participation of cytosolic phospholipase A2α and CoA-dependent acyltransferases. PI(20:4/20:4) formation is also detected in vivo in murine peritonitis exudates. Elevating the intracellular concentration of PI(20:4/20:4) by introducing the lipid into the cells results in enhancement of the microbicidal capacity of macrophages, as measured by reactive oxygen metabolite production and lysozyme release. These findings suggest that PI(20:4/20:4) is a novel bioactive inositol phospholipid molecule that regulates innate immune responses in macrophages.

Role of SHIP1 in bone biology

The bone marrow milieu comprising both hematopoietic and nonhematopoietic lineages has a unique structural organization. Bone undergoes continuous remodeling throughout life. This dynamic process involves a balance between bone-forming osteoblasts (OBs) derived from multipotent mesenchymal stem cells (MSCs) and bone-resorbing osteoclasts (OCs) derived from hematopoietic stem cells (HSCs). Src homology 2-domain-containing inositol 5'-phosphatase 1 (SHIP1) regulates cellular processes such as proliferation, differentiation, and survival via the PI3K/Akt signaling pathway initiated at the plasma membrane. SHIP1-deficient mice also exhibit profound osteoporosis that has been proposed to result from hyperresorptive activity by OCs. We have previously observed that SHIP1 is expressed in primary OBs, which display defective development in SHIP1-deficient mice. These findings led us to question whether SHIP1 plays a functional role in osteolineage development from MSC in vivo, which contributes to the osteoporotic phenotype in germline SHIP1 knockout mice. In this short review, we discuss our current understanding of inositol phospholipid signaling downstream of SHIP1 in bone biology.

Sensory receptors—design principles revisited

Sensory receptors—design principles revisited

Role of inositol phospholipid signaling in natural killer cell biology

Natural killer (NK) cells are important for host defense against malignancy and infection. At a cellular level NK cells are activated when signals from activating receptors exceed signaling from inhibitory receptors. At a molecular level NK cells undergo an education process to both prevent autoimmunity and acquire lytic capacity. Mouse models have shown important roles for inositol phospholipid signaling in lymphocytes. NK cells from mice with deletion in different members of the inositol phospholipid signaling pathway exhibit defects in development, NK cell repertoire expression and effector function. Here we review the current state of knowledge concerning the function of inositol phospholipid signaling components in NK cell biology.

GLUT4 Traffic through an ESCRT-III-Dependent Sorting Compartment in Adipocytes

In insulin target tissues, GLUT4 is known to traffic through multiple compartments that may involve ubiquitin- and/or SUMO-dependent targeting. During these trafficking steps, GLUT4 is sorted into a storage reservoir compartment that is acutely released by insulin signalling processes that are downstream of PI 3-kinase associated changes in inositol phospholipids. As ESCRT components have recently been found to influence cellular sorting processes that are related to changes in both ubiquitination and inositol phospholipids, we have examined whether GLUT4 traffic is routed through ESCRT dependent sorting steps. Introduction of the dominant negative inhibitory constructs of the ESCRT-III components CHMP3 (CHMP3(1–179)) and Vps4 (GFP-Vps4E235Q) into rat adipocytes leads to the accumulation of GLUT4 in large, coalesced and extended vesicles structures that co-localise with the inhibitory constructs over large parts of the extended structure. A new swollen hybrid and extensively ubiquitinated compartment is produced in which GLUT4 co-localises more extensively with the endosomal markers including EEA1 and transferrin receptors but also with the TGN marker syntaxin6. These perturbations are associated with failure of insulin action on GLUT4 traffic to the cell surface and suggest impairment in an ESCRT-dependent sorting step used for GLUT4 traffic to its specialised reservoir compartment.

Recent Advances in Plant Cell Nuclear Signaling

In plants, a growing amount of evidence suggests an important role for the nucleus in the establishment of developmental and adaptive processes. Recent years have contributed to an increase in the knowledge about the nuclear compartment that has been considered for a long time as an organelle in which only mechanisms such as transcription and DNA replication occurred. The scenario appears now largely more complicated, in that the nucleus has been shown to harbor a number of partially autonomous regulatory mechanisms, to such an extent that some authors claimed that the nucleus is ‘a cell within a cell’ (Rodrigues et al., 2009).

Phosphoinositide Signaling

"All things flow and change…even in the stillest matter there is unseen flux and movement." Attributed to Heraclitus (530-470 BC), from The Story of Philosophy by Will Durant. Heraclitus, a Greek philosopher, was thinking on a much larger scale than molecular signaling; however, his visionary comments are an important reminder for those studying signaling today. Even in unstimulated cells, signaling pathways are in constant metabolic flux and provide basal signals that travel throughout the organism. In addition, negatively charged phospholipids, such as the polyphosphorylated inositol phospholipids, provide a circuit board of on/off switches for attracting or repelling proteins that define the membranes of the cell. This template of charged phospholipids is sensitive to discrete changes and metabolic fluxes-e.g., in pH and cations-which contribute to the oscillating signals in the cell. The inherent complexities of a constantly fluctuating system make understanding how plants integrate and process signals challenging. In this review we discuss one aspect of lipid signaling: the inositol family of negatively charged phospholipids and their functions as molecular sensors and regulators of metabolic flux in plants.

Myo-Inositol Uptake Is Essential for Bulk Inositol Phospholipid but Not Glycosylphosphatidylinositol Synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.

Coordination of stress response signaling and phospholipid metabolism in yeast

Previously we showed that the availability of the lipid precursor inositol influences the levels and rates of synthesis and turnover of inositol phospholipids, phosphoinositides and complex sphingolipids. To discover the inositol‐dependent mechanisms that lead to activation of lipid‐mediated signaling pathways, we compared the simultaneous changes in lipid metabolism with those occurring in gene expression in yeast cells during a 3‐hour period of inositol starvation followed by a 30‐min period of inositol add‐back. We found that inositol starvation and add‐back led to dramatic changes in the abundance of inositol‐containing phospholipids, including phosphatidylinositol, the phosphoinositides, and complex sphingolipids. These changes were accompanied by the altered expression of hundreds of genes involved in diverse stress response pathways. Significantly we found that many mutants deleted for nonessential genes in the same stress response pathways exhibit inositol auxotrophy. These results suggest that activation of stress response signaling pathways is coordinated with inositol‐containing phospholipid metabolism.

The role of cytoskeleton, components of inositol phospholipid signaling pathway and iron in Ehrlichia canis in vitro proliferation

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.

A role for phosphoinositides in regulating plant nuclear functions.

Nuclear localized inositol phospholipids and inositol phosphates are important for regulating many essential processes in animal and yeast cells such as DNA replication, recombination, RNA processing, mRNA export and cell cycle progression. An overview of the current literature indicates the presence of a plant nuclear phosphoinositide (PI) pathway. Inositol phospholipids, inositol phosphates, and enzymes of the PI pathway have been identified in plant nuclei and are implicated in DNA replication, chromatin remodeling, stress responses and hormone signaling. In this review, the potential functions of the nuclear PI pathway in plants are discussed within the context of the animal and yeast literature. It is anticipated that future research will help shed light on the functional significance of the nuclear PI pathway in plants.

Chronic exposure to high fatty acids impedes receptor agonist-induced nitric oxide production and increments of cytosolic Ca2+ levels in endothelial cells

Dyslipidemia is a common metabolic disorder in diabetes. Nitric oxide (NO) production from endothelium plays the primary role in endothelium-mediated vascular relaxation and other endothelial functions. Therefore, we investigated the effects of elevated free fatty acids (FFA) on the stimulation of NO production by phospholipase C (PLC)-activating receptor agonists (potent physiological endothelium-dependent vasodilators) and defined the possible alterations of signaling pathways implicated in this scenario. Exposure of bovine aortic endothelial cells (BAECs) to high concentrations of a mixture of fatty acids (oleate and palmitate) for 5 or 10 days significantly reduced NO production evoked by receptor agonists (bradykinin or ATP) in a time- and dose-dependent manner. Such defects were not associated with alterations of either endothelial NO synthase mass or inositol phospholipid contents but were probably due to reduced elevations of intracellular free Ca(2)(+) levels ([Ca(2)(+)](i)) under these conditions. Exposure of BAECs to FFA significantly attenuated agonist-induced [Ca(2)(+)](i) increases by up to 54% in a dose- and time-dependent manner. Moreover, bradykinin receptor affinity on the cell surface was significantly decreased by high concentrations of FFA. The morphology of BAECs was altered after 10-day culture with high FFA. Co-culture with protein kinase C (PKC) inhibitors or antioxidants was able to reverse the impairments of receptor agonist-induced NO production and [Ca(2)(+)](i) rises as well as the alteration of receptor affinity in BAECs exposed to FFA. These data indicate that chronic exposure to high FFA reduces NO generation in endothelial cells probably by impairing PLC-mediated Ca(2)(+) signaling pathway through activation of PKC and excess generation of oxidants.

A Small Molecule Mimicking a Phosphatidylinositol (4,5)-Bisphosphate Binding Pleckstrin Homology Domain

Inositol phospholipids have emerged as important key players in a wide variety of cellular functions. Among the seven existing inositol phospholipids, phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) has attracted much attention in recent years due to its important role in numerous cellular signaling events and regulations, which in turn impact several human diseases. This particular lipid is recognized in the cell by specific lipid binding domains, such as the Pleckstrin-homology (PH) domain, which is also employed as a tool to monitor this important lipid. Here, we describe the synthesis and biological characterization of a small molecule that mimics the PH domain as judged by its ability to bind specifically to only PI(4,5)P(2) and effectively compete with the PH domain in vitro and in a cellular environment. The binding constant of this small molecule PH domain mimetic (PHDM) was determined to be 17.6 ± 10.1 μM, similar in potency to the PH domain. Using NIH 3T3 mouse fibroblast cells we demonstrated that this compound is cell-permeable and able to modulate PI(4,5)P(2)-dependent effects in a cellular environment such as the endocytosis of the transferrin receptor, loss of mitochondria, as well as stress fiber formation. This highly PI(4,5)P(2)-specific chemical mimetic of a PH domain not only is a powerful research tool but might also be a lead compound in future drug developments targeting PI(4,5)P(2)-dependent diseases such as Lowe syndrome.

Involvement of Protein Kinase Cδ/Extracellular Signal-regulated Kinase/Poly(ADP-ribose) Polymerase-1 (PARP-1) Signaling Pathway in Histamine-induced Up-regulation of Histamine H1 Receptor Gene Expression in HeLa Cells

The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.

Emerging roles of phosphoinositide-specific phospholipases C in the ciliates Tetrahymena and Paramecium

Phospholipases C (PLCs) that hydrolyze inositol phospholipids regulate vital cellular functions in both eukaryotic and prokaryotic organisms. The PLC superfamily consists of eukaryotic phosphoinositide-specific PLCs (PI-PLCs), bacterial PLCs and trypanosomal PLCs.1 PI-PLCs hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) to produce inositol 1,4,5-trisphosphate (Ins1,4,5P3) and constitute a hallmark feature of eukaryotic cells. In metazoa, this reaction is coupled to receptor signaling via specific PI-PLC isoforms and results in acute increase of cytosolic Ca2+ levels by Ins1,4,5P3-sensitive Ca2+ channels (IP3-receptors, IP3Rs).2 A striking result of many studies so far has been the presence of a single PI-PLC gene in all unicellular eukaryotes investigated, as opposed to expansion of PI-PLC isoforms in metazoa;3 this has suggested that a single housekeeping PI-PLC represents an archetypal and simplified form of PI-PLC signaling.3 Several studies however have noted a unique expansion of PI-PLC/IP3R pathway components in ciliates.4,5 In a recent paper we showed the presence of multiple functional PI-PLC genes in Tetrahymena thermophila and biochemical characterization, pharmacological studies and study of their expression patterns suggested that they are likely to serve distinct non-redundant roles.4 In this report we discuss these studies and how they advance our understanding of PI-PLC functions in ciliates.

Emerging roles of phosphoinositide-specific phospholipases C in the ciliates Tetrahymena and Paramecium.

Phospholipases C (PLCs) that hydrolyze inositol phospholipids regulate vital cellular functions in both eukaryotic and prokaryotic organisms. The PLC superfamily consists of eukaryotic phosphoinositide-specific PLCs (PI-PLCs), bacterial PLCs and trypanosomal PLCs.1 PI-PLCs hydrolyze phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) to produce inositol-1,4,5-trisphosphate (Ins1,4,5P(3)) and constitute a hallmark feature of eukaryotic cells. In metazoa, this reaction is coupled to receptor signaling via specific PI-PLC isoforms and results in acute increase of cytosolic Ca(2+) levels by Ins1,4,5P(3)-sensitive Ca(2+) channels (IP(3)-receptors, IP3Rs).2 A striking result of many studies so far has been the presence of a single PI-PLC gene in all unicellular eukaryotes investigated, as opposed to expansion of PI-PLC isoforms in metazoa;3 this has suggested that a single housekeeping PI-PLC represents an archetypal and simplified form of PI-PLC signaling.3 Several studies however have noted a unique expansion of PI-PLC/IP3R pathway components in ciliates.4,5 In a recent paper we showed the presence of multiple functional PI-PLC genes in Tetrahymena thermophila and biochemical characterization, pharmacological studies and study of their expression patterns suggested that they are likely to serve distinct non-redundant roles.4 In this report we discuss these studies and how they advance our understanding of PI-PLC functions in ciliates.

P.2.c.016 Decrease of bone mineral density in postmenopausal women with depressive disorder taking selective serotonin reuptake inhibitor

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603–7608) that serotonin induces: (1) a very rapid formation of PI; and (2) alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.

P.2.c.017 Creating a new clinically relevant endpoint in the PNB01 trial programme: the Early and Sustained Antidepressant Response (ESR)

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603–7608) that serotonin induces: (1) a very rapid formation of PI; and (2) alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.

Phosphoinositides in Cell Architecture

Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.