Physiological function of phospholipase D2 in anti-tumor immunity: regulation of CD8+ T lymphocyte proliferation

Two major phospholipase D (PLD) isozymes in mammals, PLD1 and PLD2, hydrolyze the membrane phospholipid phosphatidylcholine to choline and the lipid messenger phosphatidic acid. Although their roles in cancer cells have been well studied, their functions in tumor microenvironment have not yet been clarified. Here, we demonstrate that PLD2 in cytotoxic CD8+ T cells plays a crucial role in anti-tumor immunity by regulating their cell proliferation. We found that growth of tumors formed by subcutaneously transplanted cancer cells is enhanced in Pld2-knockout mice. Interestingly, this phenotype was found to be at least in part attributable to the ablation of Pld2 from bone marrow cells. The number of CD8+ T cells, which induce cancer cell death, significantly decreased in the tumor produced in Pld2-knockout mice. In addition, CD3/CD28-stimulated proliferation of primary cultured splenic CD8+ T cells is markedly suppressed by Pld2 ablation. Finally, CD3/CD28-dependent activation of Erk1/2 and Ras is inhibited in Pld2-deleted CD8+ T cells. Collectively, these results indicate that PLD2 in CD8+ T cells plays a key role in their proliferation through activation of the Ras/Erk signaling pathway, thereby regulating anti-tumor immunity

Phospholipase D2 promotes disease progression of renal cell carcinoma through the induction of angiogenin

A hallmark of clear cell renal cell carcinoma (ccRCC) is the presence of intracellular lipid droplets (LD) and it is assumed that phosphatidic acid (PA) produced by phospholipase D (PLD) plays some role in the LD formation. However, little is known about the significance of PLD in ccRCC. In this study, we examined the expression levels of PLD in ccRCC. The classical mammalian isoforms of PLD are PLD1 and PLD2, and the levels of both mRNA were higher at the primary tumor sites than in normal kidney tissues. Similarly, both PLD were significantly abundant in tumor cells as determined by analysis using immunohistochemical staining. Importantly, a higher level of PLD was significantly associated with a higher tumor stage and grade. Because PLD2 knockdown effectively suppressed the cell proliferation and invasion of ccRCC as compared with PLD1 in vitro, we examined the effect of PLD2 in vivo. Notably, shRNA‐mediated knockdown of PLD2 suppressed the growth and invasion of tumors in nude mouse xenograft models. Moreover, the higher expression of PLD2 was significantly associated with poorer prognosis in 67 patients. As for genes relating to the tumor invasion of PLD2, we found that angiogenin (ANG) was positively regulated by PLD2. In fact, the expression levels of ANG were elevated in tumor tissues as compared with normal kidney and the inhibition of ANG activity with a neutralizing antibody significantly suppressed tumor invasion. Overall, we revealed for the first time that PLD2‐produced PA promoted cell invasion through the expression of ANG in ccRCC cells

Arf6 in lymphatic endothelial cells regulates lymphangiogenesis by controlling directional cell migration

The small GTPase Arf6 plays pivotal roles in a wide variety of cellular events such as endocytosis, exocytosis, and actin cytoskeleton reorganization. However, the physiological functions of Arf6 at the whole animal level have not yet been thoroughly understood. Here, we show that Arf6 regulates developmental and tumor lymphangiogenesis in mice. Lymphatic endothelial cell (LEC)-specific Arf6 conditional knockout (LEC-Arf6 cKO) mouse embryos exhibit severe skin edema and impairment in the formation of lymphatic vessel network at the mid-gestation stage. Knockdown of Arf6 in human LECs inhibits in vitro capillary tube formation and directed cell migration induced by vascular endothelial growth factor-C (VEGF-C) by inhibiting VEGF-C-induced internalization of β1 integrin. Finally, we found that LEC-Arf6 cKO mice transplanted with B16 melanoma cells attenuated tumor lymphangiogenesis and progression. Collectively, these results demonstrate that Arf6 in LECs plays a crucial role in physiological and pathological lymphangiogenesis

Regulation of HGF-induced hepatocyte proliferation by the small GTPase Arf6 through the PIP2-producing enzyme PIP5K1A

HGF and its receptor c-Met are critical molecules in various biological processes. Others and we have previously shown that the small GTPase Arf6 plays a pivotal role in HGF signaling in hepatocytes. However, the molecular mechanism of how Arf6 regulates HGF signaling is unclear. Here, we show that Arf6 plays an important role in HGF-stimulated hepatocyte proliferation and liver regeneration through the phosphatidylinositol 4,5-bisphosphate (PIP2)-producing enzyme PIP5K1A. We find that knockdown of Arf6 and PIP5K1A in HepG2 cells inhibits HGF-stimulated proliferation, Akt activation, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and its precursor PIP2. Interestingly, PIP5K1A is recruited to c-Met upon HGF stimulation in an Arf6 activity-dependent manner. Finally, we show that hepatocyte proliferation and liver regeneration after partial hepatectomy are suppressed in Pip5k1a knockout mice. These results provide insight into the molecular mechanism for HGF-stimulated hepatocyte proliferation and liver regeneration: Arf6 recruits PIP5K1A to c-Met and activates it upon HGF stimulation to produce PIP2 and subsequently PIP3, which in turn activates Akt to promote hepatocyte proliferation, thereby accelerating liver regeneration after liver injury

The small G protein Arf6 expressed in keratinocytes by HGF stimulation is a regulator for skin wound healing

The earlier step of cutaneous wound healing process, re-epithelialization of the wounded skin, is triggered by a variety of growth factors. However, molecular mechanisms through which growth factors trigger skin wound healing are less understood. Here, we demonstrate that hepatocyte growth factor (HGF)/c-Met signaling-induced expression of the small G protein Arf6 mRNA in keratinocytes is essential for the skin wound healing. Arf6 mRNA expression was dramatically induced in keratinocytes at the wounded skin, which was specifically suppressed by the c-Met inhibitor. Wound healing of the skin was significantly delayed in keratinocyte-specific Arf6 conditional knockout mice. Furthermore, Arf6 deletion from keratinocytes remarkably suppressed HGF-stimulated cell migration and peripheral membrane ruffle formation, but did not affect skin morphology and proliferation/differentiation of keratinocytes. These results are consistent with the notion that Arf6 expressed in skin keratinocytes through the HGF/c-Met signaling pathway in response to skin wounding plays an important role in skin wound healing by regulating membrane dynamics-based motogenic cellular function of keratinocytes

Coupling Of Hydrodynamic And Wave Models For Storm Tide Simulations: A Case Study For Hurricane Floyd (1999)

This dissertation presents the development of a two-dimensional St. Johns River model and the coupling of hydrodynamic and wave models for the simulation of storm tides. The hydrodynamic model employed for calculating tides and surges is ADCIRC-2DDI (ADvanced CIRCulation Model for Shelves, Coasts and Estuaries, Two-Dimensional Depth Integrated) developed by Luettich et al. (1992). The finite element based model solves the fully nonlinear shallow water equations in the generalized wave continuity form. Hydrodynamic applications are operated with the following forcings: 1) astronomical tides, 2) inflows from tributaries, 3) meteorological effects (winds and pressure), and 4) waves (wind-induced waves). The wave model applied for wind-induced wave simulation is the third-generation SWAN (Simulating WAves Nearshore), applicable to the estimation of wave parameters in coastal areas and estuaries. The SWAN model is governed by the wave action balance equation driven by wind, sea surface elevations and current conditions (Holthuijsen et al. 2004). The overall work is comprised of three major phases: 1) To develop a model domain that incorporates the entire East Coast of the United States, Gulf of Mexico and Caribbean Sea, while honing in on the St. Johns River area; 2) To employ output from the SWAN model with the ADCIRC model and produce a uni-directional coupling of the two models in order to investigate the effects of the wave radiation stresses; 3) To couple the ADCIRC model with the SWAN model to describe the complete interactions of the two physical processes. Model calibration and comparisons are accomplished in three steps. First, astronomical tide simulation results are calibrated with historical NOS (National Ocean Service) tide data. Second, overland and riverine flows and meteorological effects are included, and computed river levels are compared with the historical NOS water level data. Finally, the storm tides generated by Hurricane Floyd are simulated and compared with historical data. This research results in a prototype for real-time simulation of tides and waves for flash flood and river-stage forecasting efforts of the NWS Forecasting Centers that border coastal areas. The following two main conclusions are reported: 1) regardless of whether one uses uni-coupling or coupling, wind-induced waves result in an approximately 10 15 % higher peak storm tide level than without any coupling; and 2) the wave-current interaction described by the coupling model results in decreasing peaks and increasing troughs in the storm tide hydrograph. Two main corollary conclusions are also drawn from a 122-day hindcast for the period spanning June 1 October 1, 2005. First, wind forcing for the St. Johns River is equal to or greater than that of astronomic tides and generally supersedes the impact of inflows, while pressure variations have a minimal impact. Secondly, water levels inside the St. Johns River depend on the wind forcings in the deep ocean; however, if one applies an elevation hydrograph boundary condition from a large-scale domain model to a local-scale domain model the results are highly accurate

脱ユビキチン化酵素TRE17によるクラスリン非依存性カーゴ蛋白質の細胞内輸送制御

科学研究費助成事業 研究成果報告書：基盤研究(C)2014-2016課題番号 : 2644004

Coupling Of Hydrodynamic And Wave Models: Case Study For Hurricane Floyd (1999) Hindcast

This paper demonstrates a practical application of coupling a hydrodynamic model with a wave model for the calculation of storm tide elevations in the St. Johns River (Northeastern Florida). Hurricane Floyd (1999) is chosen as the storm of interest due to its track which paralleled the northeast coast of Florida without making a direct landfall on the St. Johns River. The advanced circulation (ADCIRC) numerical code is applied as the hydrodynamic model for the computation of two-dimensional circulation resulting from astronomic tides and meteorologically induced storm surge. The simulating waves nearshore (SWAN) numerical code is applied as the wave model for the computation of wind-induced waves. Two model implementations are considered in order to investigate the effect of short wave contributions on the overall storm tide water level: (1) a one-way coupling procedure that transfers gradient of wave radiation stresses from SWAN to ADCIRC; and (2) a two-way coupling procedure that builds on the former to include feedback of water levels and currents from ADCIRC to SWAN. Simulated storm tide elevations are compared to historical National Ocean Service data to result in two major conclusions: (1) wind-induced waves play a significant role in the storm tide (contributing 10-15% of the peak water level), namely with respect to the transfer of momentum from the dissipation of short waves to the long-wave motion of the storm surge; and (2) a local-scale hydrodynamic model requires the application of a hydrograph boundary condition in order to account for the remote effects of the storm surge response. © 2008 ASCE