Takehiko Sasaki

Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-γ and PTEN

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol–phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-γ (PI(3)Kγ) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kγ and PTEN control electrotaxis.

Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate–dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D–mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.

Mammalian phosphoinositide kinases and phosphatases.

Phosphoinositides are lipids that are present in the cytoplasmic leaflet of a cells plasma and internal membranes and play pivotal roles in the regulation of a wide variety of cellular processes. Phosphoinositides are molecularly diverse due to variable phosphorylation of the hydroxyl groups of their inositol rings. The rapid and reversible configuration of the seven known phosphoinositide species is controlled by a battery of phosphoinositide kinases and phosphoinositide phosphatases, which are thus critical for phosphoinositide isomer-specific localization and functions. Significantly, a given phosphoinositide generated by different isozymes of these phosphoinositide kinases and phosphatases can have different biological effects. In mammals, close to 50 genes encode the phosphoinositide kinases and phosphoinositide phosphatases that regulate phosphoinositide metabolism and thus allow cells to respond rapidly and effectively to ever-changing environmental cues. Understanding the distinct and overlapping functions of these phosphoinositide-metabolizing enzymes is important for our knowledge of both normal human physiology and the growing list of human diseases whose etiologies involve these proteins. This review summarizes the structural and biological properties of all the known mammalian phosphoinositide kinases and phosphoinositide phosphatases, as well as their associations with human disorders.

A voltage-sensing phosphatase, Ci-VSP, which shares -sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol lipids play diverse physiological roles, and their concentrations are tightly regulated by various kinases and phosphatases. The enzymatic activity of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP), recently identified as a member of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) family of phosphatidylinositol phosphatases, is regulated by its own voltage-sensor domain in a voltage-dependent manner. However, a detailed mechanism of Ci-VSP regulation and its substrate specificity remain unknown. Here we determined the in vitro substrate specificity of Ci-VSP by measuring the phosphoinositide phosphatase activity of the Ci-VSP cytoplasmic phosphatase domain. Despite the high degree of identity shared between the active sites of PTEN and Ci-VSP, Ci-VSP dephosphorylates not only the PTEN substrate, phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], but also, unlike PTEN, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Enzymatic action on PI(4,5)P2 removes the phosphate at position 5 of the inositol ring, resulting in the production of phosphatidylinositol 4-phosphate [PI(4)P]. The active site Cys-X5-Arg (CX5R) sequence of Ci-VSP differs with that of PTEN only at amino acid 365 where a glycine residue in Ci-VSP is replaced by an alanine in PTEN. Ci-VSP with a G365A mutation no longer dephosphorylates PI(4,5)P2 and is not capable of inducing depolarization-dependent rundown of a PI(4,5)P2-dependent potassium channel. These results indicate that Ci-VSP is a PI(3,4,5)P3/PI(4,5)P2 phosphatase that uniquely functions in the voltage-dependent regulation of ion channels through regulation of PI(4,5)P2 levels.

Involvement of Proton-Sensing TDAG8 in Extracellular Acidification-Induced Inhibition of Proinflammatory Cytokine Production in Peritoneal Macrophages

Extracellular acidification inhibited LPS-induced TNF-α protein production, which was associated with an inhibition of TNF-α mRNA expression, in mouse peritoneal macrophages. The LPS-induced cytokine production was also inhibited by Gs protein-coupled receptor agonists prostaglandin E1 and isoproterenol. Among OGR1 family proton-sensing GTP-binding regulatory protein-coupled receptors, TDAG8, OGR1, and G2A are expressed in the cells. The inhibitory action by acidic pH on TNF-α production was significantly attenuated in macrophages from TDAG8Tp/Tp mice but not in those from OGR1geo/geo mice. Moreover, small interfering RNA specific to TDAG8, but not to G2A, clearly attenuated the acidification-induced inhibition of TNF-α production. On the other hand, the down-regulation or deficiency of TDAG8 hardly affected prostaglandin E1- or isoproterenol-induced actions. LPS-induced IL-6 production was also inhibited by extracellular acidification in a manner that was sensitive to TDAG8 expression. The acidic pH-induced inhibitory action on the cytokine production was significantly reversed either by a small interfering RNA specific to Gs proteins or by a protein kinase A (PKA)-specific inhibitor H89. Indeed, a PKA-specific cAMP derivative inhibited LPS-induced cytokine production. Moreover, acidification induced cAMP accumulation in a TDAG8-specific way. We conclude that TDAG8, at least partly, mediates the extracellular acidification-induced inhibition of proinflammatory cytokine production through the Gs protein/cAMP/PKA signaling pathway in mouse macrophages.

Hyperplasia and Carcinomas in Pten-Deficient Mice and Reduced PTEN Protein in Human Bladder Cancer Patients

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate an urothelium-specific null mutation of Pten in mice [FabpCrePten(flox/flox) (FPten(flox/flox)) mice]. Histologic examination revealed that all FPten(flox/flox) mice exhibited urothelial hyperplasia in which component cells showed enlarged nuclei and increased cell size. With time, 10% of FPten(flox/flox) mice spontaneously developed pedicellate papillary transitional cell carcinomas (TCC). This type of tumor also arose in FPten(flox/flox) mice treated with the chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. FPten(flox/flox) urothelial cells were hyperproliferative and showed increased activation of the survival signaling molecules Akt and extracellular signal-regulated kinase. In humans, 53% of primary bladder cancer patients exhibited decreased or absent expression of PTEN protein in either the cytoplasm or nucleus of tumor cells. In early bladder cancers, PTEN expression was repressed in 42% of superficial papillary TCC but in only 8% of cases of carcinoma in situ (CIS). In advanced bladder cancers, PTEN protein was significantly reduced (particularly in the nucleus) in 94% of cases, and this decrease in PTEN correlated with disease stage and grade. Thus, PTEN deficiency may contribute to bladder cancer both by initiating superficial papillary TCC and by promoting the progression of CIS to advanced invasive and metastatic forms.

Eicosapentaenoic acid ameliorates steatohepatitis and hepatocellular carcinoma in hepatocyte-specific Pten-deficient mice☆

BACKGROUND/AIMSnEicosapentaenoic acid (EPA) has been known as a reagent for improving lipid metabolism and inflammation. Hepatocyte-specific Pten-deficient mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). Therefore, we administered EPA to Pten-deficient mice to investigate the mechanisms of NASH.nnnMETHODSnPten-deficient mice were assigned to a control group fed with a standard chow or an EPA group fed with a 5% EPA-supplemented standard chow. At 40 weeks, livers from each group were processed to measure triglyceride content, gene expression analysis, Western blotting analysis, and histological examination. Level of serum reactive oxygen species (ROS) was also determined. Forty- and 76-week-old mice were used in tumor burden experiments.nnnRESULTSnEPA-ameliorated hepatic steatosis in Pten-deficient mice was based on decreased expression of AMPKalpha1-mediated SREBP-1c and increased PPARalpha expression. The EPA group exhibited less severe chronic hepatic inflammation compared to the control group, resulting from decreased ROS formation and a dramatically low ratio of arachidonic acid to EPA. Moreover, EPA inhibited development of hepatocellular carcinoma (HCC) in Pten-deficient mice based on an inhibition of MAPK activity and a low ratio of oleic to stealic acid, and a reduction in ROS formation.nnnCONCLUSIONSnEPA ameliorated steatohepatitis and development of HCC in Pten-deficient mice.

Crucial role of the small GTPase ARF6 in hepatic cord formation during liver development.

ABSTRACT The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6−/− embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6−/− fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.

Loss of PIP5KIγ, unlike other PIP5KI isoforms, impairs the integrity of the membrane cytoskeleton in murine megakaryocytes

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is an abundant phospholipid that contributes to second messenger formation and has also been shown to contribute to the regulation of cytoskeletal dynamics in all eukaryotic cells. Although the alpha, beta, and gamma isoforms of phosphatidylinositol-4-phosphate-5-kinase I (PIP5KI) all synthesize PIP2, mammalian cells usually contain more than one PIP5KI isoform. This raises the question of whether different isoforms of PIP5KI fulfill different functions. Given the speculated role of PIP(2) in platelet and megakaryocyte actin dynamics, we analyzed murine megakaryocytes lacking individual PIP5KI isoforms. PIP5KIgamma(-/-) megakaryocytes exhibited plasma membrane blebbing accompanied by a decreased association of the membrane with the cytoskeleton. This membrane defect was rescued by adding back wild-type PIP5KIgamma, but not by adding a catalytically inactive mutant or a splice variant lacking the talin-binding motif. Notably, both PIP5KIbeta- and PIP5KIgamma(-/-) cells had impaired PIP(2) synthesis. However, PIP5KIbeta-null cells lacked the membrane-cytoskeleton defect. Furthermore, overexpressing PIP5KIbeta in PIP5KIgamma(-/-) cells failed to revert this defect. Megakaryocytes lacking the PIP5KIgamma-binding partner, talin1, mimicked the membrane-cytoskeleton defect phenotype seen in PIP5KIgamma(-/-) cells. These findings demonstrate a unique role for PIP5KIgamma in the anchoring of the cell membrane to the cytoskeleton in megakaryocytes, probably through a pathway involving talin. These observations further demonstrate that individual PIP5KI isoforms fulfill distinct functions within cells.

Allergic airway hyperresponsiveness, inflammation, and remodeling do not develop in phosphoinositide 3-kinase γ–deficient mice

BACKGROUNDnBronchial asthma is characterized by chronic airway inflammation caused by inflammatory cells. Phosphoinositide 3-kinases (PI3Ks) are known to play a prominent role in fundamental cellular responses of various inflammatory cells, including proliferation, differentiation, and cell migration. PI3Ks therefore are expected to have therapeutic potential for asthma. Although some investigations of the involvement between the pathogenesis of asthma and PI3K have been performed, it is unknown whether PI3Kgamma, a PI3K isoform, is involved in the pathogenesis of asthma.nnnOBJECTIVEnWe investigated the role of PI3Kgamma in allergen-induced allergic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling with PI3Kgamma-deficient mice.nnnMETHODSnAfter ovalbumin (OVA) sensitization, wild-type (WT) and PI3Kgamma-deficient mice were exposed to aerosolized OVA 3 days per week for 5 weeks.nnnRESULTSnIn OVA-sensitized and OVA-challenged (OVA/OVA) PI3Kgamma-deficient mice, levels of airway inflammation, AHR, and airway remodeling were significantly decreased compared with those in OVA/OVA WT mice. On the other hand, no significant differences were detected in serum OVA-specific IgE and IgG1 levels and CD4/CD8 balance in bronchoalveolar lavage fluid between OVA/OVA WT mice and OVA/OVA PI3Kgamma-deficient mice. To determine in which phase of allergic responses PI3Kgamma plays a role, we transferred splenocytes from OVA-sensitized WT or PI3Kgamma-deficient mice to naive mice of either genotype. Similar increased levels of eosinophils were induced in both WT recipient mice but not in both PI3Kgamma-deficient recipient mice.nnnCONCLUSIONnPI3Kgamma might be involved in allergic airway inflammation, AHR, and airway remodeling by regulating the challenge/effector phase of allergic responses.