Tsunaki Hongu

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.

Role of activation of PIP5Kγ661 by AP‐2 complex in synaptic vesicle endocytosis

Synaptic vesicles (SVs) are retrieved by clathrin‐mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP‐2 complex directly interacts with phosphatidylinositol 4‐phosphate 5‐kinase γ661 (PIP5Kγ661), the major PI(4,5)P2‐producing enzyme in the brain. The β2 subunit of AP‐2 was found to bind to the C‐terminal tail of PIP5Kγ661 and cause PIP5Kγ661 activation. The interaction is regulated by PIP5Kγ661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kγ661 C‐terminal region in hippocampal neurons suppresses depolarization‐dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kγ661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.

Key Roles for the Lipid Signaling Enzyme Phospholipase D1 in the Tumor Microenvironment During Tumor Angiogenesis and Metastasis

Genetic ablation or pharmacological inhibition of a lipid signaling enzyme attenuates tumor growth and metastasis. Targeting the Tumor Microenvironment Phospholipase D (PLD) is an enzyme that produces the signaling lipid phosphatidic acid and promotes the proliferation, survival, invasion, and metastasis of cancer cells. Chen et al. examined the role of the PLD isoforms PLD1 and PLD2 in the tumor environment. They found that compared to wild-type mice, mice deficient in PLD1 developed smaller, less vascularized tumors as well as fewer lung metastases in a xenograft model. Furthermore, mice treated with an inhibitor of PLD1 also developed smaller tumors and fewer metastases. These results suggest that PLD1 inhibitors could be developed to treat cancer. Angiogenesis inhibitors, which target tumor cells, confer only short-term benefits on tumor growth. We report that ablation of the lipid signaling enzyme phospholipase D1 (PLD1) in the tumor environment compromised the neovascularization and growth of tumors. PLD1 deficiency suppressed the activation of Akt and mitogen-activated protein kinase signaling pathways by vascular endothelial growth factor in vascular endothelial cells, resulting in decreased integrin-dependent cell adhesion to, and migration on, extracellular matrices, as well as reduced tumor angiogenesis in a xenograft model. In addition, mice lacking PLD1 incurred fewer lung metastases than did wild-type mice. Bone marrow transplantation and binding studies identified a platelet-derived mechanism involving decreased tumor cell–platelet interactions, in part because of impaired activation of αIIbβ3 integrin in platelets, which decreased the seeding of tumor cells into the lung parenchyma. Treatment with a small-molecule inhibitor of PLD1 phenocopied PLD1 deficiency, efficiently suppressing both tumor growth and metastasis in mice. These findings reveal that PLD1 in the tumor environment promotes tumor growth and metastasis and, taken together with previous reports on the roles of PLD in tumor cell–intrinsic adaptations to stress, suggest the potential use of PLD inhibitors as cancer therapeutics.

Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization, Macrophage Phagocytosis, and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.

Molecular Mechanisms of N-Formyl-Methionyl-Leucyl-Phenylalanine-Induced Superoxide Generation and Degranulation in Mouse Neutrophils: Phospholipase D Is Dispensable

ABSTRACT Phospholipase D (PLD), which produces the lipid messenger phosphatidic acid (PA), has been implicated in superoxide generation and degranulation in neutrophils. The basis for this conclusion is the observation that primary alcohols, which interfere with PLD-catalyzed PA production, inhibit these neutrophil functions. However, off-target effects of primary alcohols cannot be totally excluded. Here, we generated PLD−/− mice in order to reevaluate the involvement of PLD in and investigate the molecular mechanisms of these neutrophil functions. Surprisingly, N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced these functions in PLD−/− neutrophils, and these functions were suppressed by ethanol. These results indicate that PLD is dispensable for these neutrophil functions and that ethanol nonspecifically inhibits them, warning against the use of primary alcohols as specific inhibitors of PLD-mediated PA formation. The calcium ionophore ionomycin and the membrane-permeative compound 1-oleoyl-2-acetyl-sn-glycerol (OADG) synergistically induced superoxide generation. On the other hand, ionomycin alone induced degranulation, which was further augmented by OADG. These results demonstrate that conventional protein kinase C (cPKC) is crucial for superoxide generation, and a Ca2+-dependent signaling pathway(s) and cPKC are involved in degranulation in mouse neutrophils.

Phosphatidic acid-dependent recruitment and function of the Rac activator DOCK1 during dorsal ruffle formation.

Background: DOCK1 is an atypical Rac activator. Results: Activation of the PDGF receptor induces DOCK1 translocation to the dorsal ruffles through association with phosphatidic acid. Blocking of this interaction impairs dorsal, but not peripheral, ruffle formation. Conclusion: Phosphatidic acid acts as a lipid anchor for DOCK1 during dorsal ruffle formation. Significance: A novel regulatory mechanism for dorsal ruffle formation was identified. Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.

Arf6 regulates tumour angiogenesis and growth through HGF-induced endothelial β1 integrin recycling

Anti-angiogenic drugs targeting vascular endothelial cell growth factor receptor have provided modest clinical benefit, in part, owing to the actions of additional angiogenic factors that stimulate tumour neoangiogenesis in parallel. To overcome this redundancy, approaches targeting these other signalling pathways are required. Here we show, using endothelial cell-targeted mice, that the small GTPase Arf6 is required for hepatocyte growth factor (HGF)-induced tumour neoangiogenesis and growth. Arf6 deletion from endothelial cells abolishes HGF-stimulated β1 integrin recycling. Pharmacological inhibition of the Arf6 guanine nucleotide exchange factor (GEF) Grp1 efficiently suppresses tumour vascularization and growth. Grp1 as well as other Arf6 GEFs, such as GEP100, EFA6B and EFA6D, regulates HGF-stimulated β1 integrin recycling. These findings provide insight into the mechanism of HGF-induced tumour angiogenesis and offer the possibility that targeting the HGF-activated Arf6 signalling pathway may synergize with existing anti-angiogenic drugs to improve clinical outcomes.

Activation machinery of the small GTPase Arf6.

The small GTPase ADP-ribosylation factor 6 (Arf6) plays pivotal roles in a wide variety of cellular events, including exocytosis, endocytosis, actin cytoskeleton reorganization and phosphoinositide metabolism, in various types of cells. To control such a wide variety of actions of Arf6, activation of Arf6 could be precisely controlled by its activators, guanine nucleotide exchange factors (GEFs), in spatial and temporal manners. In this manuscript, we summarize and discuss the characteristics of previously identified GEFs specific to Arf6 and activation machineries of Arf6.

Tissue- and development-dependent expression of the small GTPase Arf6 in mice

The small GTPase Arf6 is a member of the Arf (ADP‐ribosylation factor) family. Although the function of Arf6 has been heavily studied at the cellular level, its physiological function at the whole animal level is largely unknown. In this study, we examined both the tissue distribution and developmental timing of Arf6 expression in wild type mice to obtain valuable information to speculate on the physiological function of Arf6. Western blot analysis using anti‐Arf6 antibody revealed that Arf6 was ubiquitously expressed with its developmental timing differing in a tissue‐specific manner. These results were supported by Arf6 mRNA in situ hybridization experiments, which showed that Arf6 was highly expressed in the polarized epithelial cells and embryonic mesenchymal cells of most tissues in a temporally dependent manner. Taken in toto, our results suggest that the expression of Arf6 in mouse tissues is precisely regulated in a development‐ and tissue‐dependent manner. Developmental Dynamics 239:3416–3435, 2010.

PKN3 is the major regulator of angiogenesis and tumor metastasis in mice

PKN, a conserved family member related to PKC, was the first protein kinase identified as a target of the small GTPase Rho. PKN is involved in various functions including cytoskeletal arrangement and cell adhesion. Furthermore, the enrichment of PKN3 mRNA in some cancer cell lines as well as its requirement in malignant prostate cell growth suggested its involvement in oncogenesis. Despite intensive research efforts, physiological as well as pathological roles of PKN3 in vivo remain elusive. Here, we generated mice with a targeted deletion of PKN3. The PKN3 knockout (KO) mice are viable and develop normally. However, the absence of PKN3 had an impact on angiogenesis as evidenced by marked suppressions of micro-vessel sprouting in ex vivo aortic ring assay and in vivo corneal pocket assay. Furthermore, the PKN3 KO mice exhibited an impaired lung metastasis of melanoma cells when administered from the tail vein. Importantly, PKN3 knock-down by small interfering RNA (siRNA) induced a glycosylation defect of cell-surface glycoproteins, including ICAM-1, integrin β1 and integrin α5 in HUVECs. Our data provide the first in vivo genetic demonstration that PKN3 plays critical roles in angiogenesis and tumor metastasis, and that defective maturation of cell surface glycoproteins might underlie these phenotypes.