Chisato Tanaka

Rab27a Negatively Regulates Phagocytosis by Prolongation of the Actin-coating Stage around Phagosomes

Rab27a, a Rab family small GTPase, is involved in the exocytosis of secretory granules in melanocytes and cytotoxic T-cells. Rab27a mutations cause type 2 Griscelli syndrome, which is characterized by immunodeficiency, including uncontrolled macrophage activation known as hemophagocytic syndrome. However, the role of Rab27a in phagocytosis remains elusive. Here, using macrophage-like differentiated HL-60 cells and C3bi-opsonized zymosan as a pathogen-phagocyte model, we show that Rab27a negatively regulates complement-mediated phagocytic activity in association with F-actin remodeling. We found that transfection of Rab27a shRNA into HL-60 cells enhances complement-mediated phagocytosis. To clarify the mechanisms underlying the elevated phagocytosis in Rab27a knockdown cells, we analyzed the process of phagosome formation focusing on F-actin dynamics: F-actin assembly, followed by F-actin extension around the particles and the subsequent degradation of F-actin, leading to internalization of the particles enclosed in phagosomes. Microscopic analysis revealed that these actin-related processes, including F-actin coating and F-actin degradation, proceed more rapidly in Rab27a knockdown cells than in control HL-60 cells. Both elevated phagocytosis and accelerated F-actin remodeling were restored by expression of rescue-Rab27a and Rab27a-Q78L (GTP-bound form), but not by Rab27a-T23N (GDP-bound form). Furthermore, an increased accumulation of Coronin 1A surrounding F-actin coats was observed in Rab27a knockdown cells, suggesting that the function of Coronin 1A is related to the regulation of the F-actin coating. Our findings demonstrate that Rab27a plays a direct regulatory role in the nascent process of phagocytosis by prolongation of the stage of actin coating via suppression of Coronin 1A. This study may contribute to an explanation of the underlying mechanisms of excessive phagocytosis observed in Griscelli syndrome.

Rab27a Is Essential for the Formation of Neutrophil Extracellular Traps (NETs) in Neutrophil-Like Differentiated HL60 Cells

Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs). NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS) is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA)- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an important role in NET formation induced by both Candida albicans infection and PMA treatment by regulating ROS production.

ATP‐induced osteoclast function: the formation of sealing‐zone like structure and the secretion of lytic granules via microtubule‐deacetylation under the control of Syk

Osteoclasts are bone‐resorbing cells which play an exclusive role in bone remodeling, but the molecular mechanisms of osteolysis, how osteoclasts are activated and how the lytic granules are finally released towards the bone matrix are poorly understood. Here we show that an energy molecule ATP induces osteolysis via P2X7‐nucleotide receptor and that deacetylation of α‐tubulin is essential for the whole process of osteolysis under the control of a tyrosine kinase Syk. By developing a traceable and reproducible in vitro analyzing system for osteoclast function, we found that ATP‐signaling gives rise to two events simultaneously (i) cytoskeletal reorganization for the formation of sealing zones, ring‐like adhesion structures which delimit the contact surface, and (ii) the delivery and secretion of lytic granules towards the delimited site on the matrix. We further found that deacetylation of α‐tubulin is a critical reaction for osteoclast function. Pharmacological inhibition of α‐tubulin deacetylation resulted in (i) failure of the sealing‐zone like structure formation and (ii) ceased secretion of lytic granules. Additionally, kinetics of deacetylation was found to be regulated by Syk. These data suggest a novel P2X7 microtubular regulation pathway related to Syk for a therapeutic target in osteolytic diseases.

Rab27b regulates c-kit expression by controlling the secretion of stem cell factor.

Rab27b, a subfamily of Rab27 small GTPases, was originally identified in platelets. However, the role of Rab27b in megakaryocytic lineage cells remains unknown. Here, using a human megakaryoblastic cell line, CMK, we show that Rab27b negatively regulates c-kit-expression. We found that transfection of shRNA-Rab27b into CMK cells led to specific increase in the amount of the receptor-type tyrosine kinase c-kit. To elucidate the molecular mechanisms by which Rab27b regulates c-kit expression, we analyzed the dynamics of c-kit by the stimulation with its ligand, stem cell factor (SCF). We found that cell surface expression of c-kit was promptly reduced and rapidly degraded in both CMK and Rab27b-knockdown CMK cells. Pretreatment with a lysosome inhibitor bafilomycin suppressed the degradation of c-kit, indicating that c-kit expression is controlled by SCF-induced endolysosomal degradation system. We therefore focused on the potential involvement of SCF in Rab27b-mediated effects on c-kit expression levels. We found that autocrine secretion of SCF was downregulated in Rab27b-knockdown cells as compared with parental CMK cells. These results suggest that Rab27b negatively regulates the cell surface expression of c-kit via secretion of SCF and that ligation of SCF leads to the endolysosomal degradation system of c-kit.