Person:

Williams, David

Efficient clearance of apoptotic cells by phagocytes (efferocytosis) is critical for normal tissue homeostasis and regulation of the immune system. Apoptotic cells are recognized by a vast repertoire of receptors on macrophage that lead to transient formation of phosphatidylinositol-3,4,5-trisphosphate ([PtdIns(3,4,5)P_3]) and subsequent cytoskeletal reorganization necessary for engulfment. Certain PI3K isoforms are required for engulfment of apoptotic cells, but relatively little is known about the role of lipid phosphatases in this process. In this study, we report that the activity of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatidylinositol 3-phosphatase, is elevated upon efferocytosis. Depletion of PTEN in macrophage results in elevated (PtdIns(3,4,5)P_3) production and enhanced phagocytic ability both in vivo and in vitro, whereas overexpression of wild-type PTEN abrogates this process. Loss of PTEN in macrophage leads to activation of the pleckstrin homology domain-containing guanine-nucleotide exchange factor Vav1 and subsequent activation of Rac1 GTPase, resulting in increased amounts of F-actin upon engulfment of apoptotic cells. PTEN disruption also leads to increased production of anti-inflammatory cytokine IL-10 and decreased production of proinflammatory IL-6 and TNF-α upon engulfment of apoptotic cells. These data suggest that PTEN exerts control over efferocytosis potentially by regulating (PtdIns(3,4,5)P_3) levels that modulate Rac GTPase and F-actin reorganization through Vav1 exchange factor and enhancing apoptotic cell-induced anti-inflammatory response.

Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted (Vav1^{−/−}) HSPC showed impaired early localization near (nestin^+) perivascular mesenchymal stem cells; only 6.25% of (Vav1^{−/−}) HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a (nestin^+) cell. Abnormal perivascular localization correlated with decreased retention of (Vav1^{−/−}) HSPC in the bone marrow (44–60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of (Vav1^{−/−}) cells vs. 53–63% for wild-type cells). The engraftment defect of (Vav1^{−/−}) HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, (Vav1^{−/−}) HSPC showed impaired responses to (SDF1\alpha), including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for (SDF1\alpha)-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment.