RESUMO
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.
Assuntos
Actinas/metabolismo , Macrófagos/enzimologia , Fagocitose , Fagossomos/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/genética , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/metabolismo , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Linfo-Histiocitose Hemofagocítica , Ativação de Macrófagos/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação de Sentido Incorreto , Fagossomos/genética , Piebaldismo/enzimologia , Piebaldismo/genética , Doenças da Imunodeficiência Primária , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
KIT is a receptor tyrosine kinase that is functionally relevant for hematopoiesis, mast cell development and function, gametogenesis and melanogenesis. Normal KIT signaling requires binding to stem cell factor, and PI3K-Akt is one of the putative effector pathways. In humans, germline loss-of-function KIT mutations have been associated with piebaldism - an autosomal dominant condition characterized by depigmented patches of skin and hair. Gain-of-function KIT mutations are usually acquired and have been associated with myeloid malignancies including core binding factor acute myeloid leukemia and systemic mastocytosis (SM), germ cell tumors, gastrointestinal stromal tumors and sinonasal T cell lymphomas. KITD816V is the most prevalent KIT mutation in mast cell disease and occurs in more than 90% of the cases that fulfill the World Health Organization diagnostic criteria for SM. However, its precise pathogenetic contribution is not well understood. In clinical practice, SM is considered either indolent or aggressive depending on the respective absence or presence of symptomatic target organ dysfunction aside from skin disease. In general, conventional therapy for SM is suboptimal, and efforts are under way to develop and employ small molecule drugs that target mutant KIT.