title: Involvement of protein kinase D in uridine diphosphate-induced microglial macropinocytosis and phagocytosis.
abstract: The clearance of tissue debris by microglia is a crucial component of maintaining brain homeostasis. Microglia continuously survey the brain parenchyma and utilize extracellular nucleotides to trigger the initiation of their dynamic responses. Extracellular uridine diphosphate (UDP), which leaks or is released from damaged neurons, has been reported to stimulate the phagocytotic activity of microglia through P2Y(6) receptor activation. However, the intracellular mechanisms underlying microglial P2Y(6) receptor signals have not been identified. In this study, we demonstrated that UDP stimulation induced immediate and long-lasting dynamic movements in the cell membrane. After 60 min of UDP stimulation, there was an upregulation in the number of large vacuoles formed in the cell that incorporate extracellular fluorescent-labeled dextran, which indicates microglial macropinocytosis. In addition, UDP-induced vacuole formation and continuous membrane motility were suppressed by the protein kinase D (PKD) inhibitors, Gö6976 and CID755673, unlike Gö6983, which is far less sensitive to PKD. The inhibition of PKD also reduced UDP-induced incorporation of fluorescent-labeled dextran and soluble β-amyloid and phagocytosis of microspheres. UDP induced rapid phosphorylation and membrane translocation of PKD, which was abrogated by the inhibition of protein kinase C (PKC) with Gö6983. However, Gö6983 failed to suppress UDP-induced incorporation of microspheres. Finally, we found that inhibition of PKD by CID755673 significantly suppressed UDP-induced engulfment of IgG-opsonized microspheres. These data suggest that a PKC-independent function of PKD regulates UDP-induced membrane movement and contributes to the increased uptake of extracellular fluid and microspheres in microglia.
authors.affiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi, Fukuoka, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Uesugi
authors.forename: Ayako
authors.initials: A
authors.lastname: Kataoka
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tozaki-Saitoh
authors.forename: Yui
authors.initials: Y
authors.lastname: Koga
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsuda
authors.forename: Bernard
authors.initials: B
authors.lastname: Robaye
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Boeynaems
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Inoue
mesh_terms: D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001572:Benzofurans / Q000494:pharmacology; D002478:Cells, Cultured; D017628:Microglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010873:Pinocytosis / Q000187:drug effects* / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014530:Uridine Diphosphate / Q000494:pharmacology*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
chemical_list: D001381:Azepines; D001572:Benzofurans; C534615:CID755673; D014530:Uridine Diphosphate; C089212:protein kinase D; D011493:Protein Kinase C