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1.
Berdyshev EV Gorshkova I Usatyuk P Kalari S Zhao Y Pyne NJ Pyne S Sabbadini RA Garcia JG Natarajan V 《PloS one》2011,6(1):e16571
Background
Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P1 receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.Methodology/Principal Findings
Inhibition of SphK activity with a SphK inhibitor 2-(p-Hydroxyanilino)-4-(p-Chlorophenyl) Thiazole or down-regulation of Sphk1, but not SphK2, with siRNA decreased S1Pint, and attenuated S1Pext or serum-induced motility of HPAECs. On the contrary, inhibition of S1PL with 4-deoxypyridoxine or knockdown of S1PL with siRNA increased S1Pint and potentiated motility of HPAECs to S1Pext or serum. S1Pext mediates cell motility through activation of Rac1 and IQGAP1 signal transduction in HPAECs. Silencing of SphK1 by siRNA attenuated Rac1 and IQGAP1 translocation to the cell periphery; however, knockdown of S1PL with siRNA or 4-deoxypyridoxine augmented activated Rac1 and stimulated Rac1 and IQGAP1 translocation to cell periphery. The increased cell motility mediated by down-regulation was S1PL was pertussis toxin sensitive suggesting “inside-out” signaling of intracellularly generated S1P. Although S1P did not accumulate significantly in media under basal or S1PL knockdown conditions, addition of sodium vanadate increased S1P levels in the medium and inside the cells most likely by blocking phosphatases including lipid phosphate phosphatases (LPPs). Furthermore, addition of anti-S1P mAb to the incubation medium blocked S1Pext or 4-deoxypyridoxine-dependent endothelial cell motility.Conclusions/Significance
These results suggest S1Pext mediated endothelial cell motility is dependent on intracellular S1P production, which is regulated, in part, by SphK1 and S1PL. 相似文献2.
Mirzapoiazova T Kolosova I Usatyuk PV Natarajan V Verin AD 《American journal of physiology. Lung cellular and molecular physiology》2006,291(4):L718-L724
Increased endothelial permeability is involved in the pathogenesis of many cardiovascular and pulmonary diseases. Vascular endothelial growth factor (VEGF) is a permeability-increasing cytokine. At the same time, VEGF is known to have a beneficial effect on endothelial cells (EC), increasing their survival. Pulmonary endothelium, particularly, may be exposed to higher VEGF concentrations, since the VEGF level is the higher in the lungs than in any other organ. The purpose of this work was to evaluate the effects of VEGF on barrier function and motility of cultured human pulmonary EC. Using transendothelial resistance measurements as an indicator of permeability, we found that 10 ng/ml VEGF significantly improved barrier properties of cultured human pulmonary artery EC (118.6+/-0.6% compared with 100% control, P<0.001). In contrast, challenge with 100 ng/ml VEGF decreased endothelial barrier (71.6+/-1.0% compared with 100% control, P<0.001) and caused disruption of adherens junctions. VEGF at both concentrations increased cellular migration; however, 10 ng/ml VEGF had a significantly stronger effect. VEGF caused a dose-dependent increase in intracellular Ca2+ concentration; however, phosphorylation of myosin light chain was detectably elevated only after treatment with 100 ng/ml. In contrast, 10 ng/ml but not 100 ng/ml VEGF caused a significant increase in intracellular cAMP (known barrier-protective stimulus) compared with nonstimulated cells (1,096+/-157 and 610+/-86 fmol/mg, respectively; P<0.024). Y576-specific phosphorylation of focal adhesion kinase was also stimulated by 10 ng/ml VEGF. Our data suggest that, depending on its concentration, VEGF may cause diverse effects on pulmonary endothelial permeability via different signaling pathways. 相似文献
3.
Cummings R Parinandi N Wang L Usatyuk P Natarajan V 《Molecular and cellular biochemistry》2002,(1-2):99-109
Phospholipase D (PLD), a phospholipid phosphohydrolase, catalyzes the hydrolysis of phosphatidylcholine and other membrane phospholipids to phosphatidic acid (PA) and choline. PLD, ubiquitous in mammals, is a critical enzyme in intracellular signal transduction. PA generated by agonist- or reactive oxygen species (ROS)-mediated activation of the PLDI and PLD2 isoforms can be subsequently converted to lysoPA (LPA) or diacylglycerol (DAG) by phospholipase A1/A2 or lipid phosphate phosphatases. In pulmonary epithelial and vascular endothelial cells, a wide variety of agonists stimulate PLD and involve Src kinases, p-38 mitogen activated protein kinase, calcium and small G proteins. PA derived from the PLD pathway has second-messenger functions. In endothelial cells, PA regulates NAD[P]H oxidase activity and barrier function. In airway epithelial cells, sphingosine-1-phosphate and PA-induced IL-8 secretion and ERKI/2 phosphorylation is regulated by PA. PA can be metabolized to LPA and DAG, which function as first- and second-messengers, respectively. Signaling enzymes such as Raf 1, protein kinase Czeta and type I phosphatidylinositol-4-phosphate 5-kinase are also regulated by PA in mammalian cells. Thus, PA and its metabolic products play a central role in modulating endothelial and epithelial cell functions. 相似文献
4.
Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction 总被引:3,自引:0,他引:3
Birukova AA Birukov KG Adyshev D Usatyuk P Natarajan V Garcia JG Verin AD 《Journal of cellular physiology》2005,204(3):934-947
Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling. 相似文献
5.
Cummings Rhett Parinandi Narasimham Wang Lixin Usatyuk Peter Natarajan Viswanathan 《Molecular and cellular biochemistry》2002,(1):99-109
Phospholipase D (PLD), a phospholipid phosphohydrolase, catalyzes the hydrolysis of phosphatidylcholine and other membrane phospholipids to phosphatidic acid (PA) and choline. PLD, ubiquitous in mammals, is a critical enzyme in intracellular signal transduction. PA generated by agonist- or reactive oxygen species (ROS)-mediated activation of the PLD1 and PLD2 isoforms can be subsequently converted to lysoPA (LPA) or diacylglycerol (DAG) by phospholipase A1/A2 or lipid phosphate phosphatases. In pulmonary epithelial and vascular endothelial cells, a wide variety of agonists stimulate PLD and involve Src kinases, p-38 mitogen activated protein kinase, calcium and small G proteins. PA derived from the PLD pathway has second messenger functions. In endothelial cells, PA regulates NAD[P]H oxidase activity and barrier function. In airway epithelial cells, sphingosine-1-phosphate and PA-induced IL-8 secretion and ERK1/2 phosphorylation is regulated by PA. PA can be metabolized to LPA and DAG, which function as first- and second-messengers, respectively. Signaling enzymes such as Raf 1, protein kinase C and type I phosphatidylinositol-4-phosphate 5-kinase are also regulated by PA in mammalian cells. Thus, PA and its metabolic products play a central role in modulating endothelial and epithelial cell functions. 相似文献
6.
Chowdhury AK Watkins T Parinandi NL Saatian B Kleinberg ME Usatyuk PV Natarajan V 《The Journal of biological chemistry》2005,280(21):20700-20711
Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs. 相似文献
7.
Peter V. Usatyuk Irina A. Gorshkova Donghong He Yutong Zhao Satish K. Kalari Joe G. N. Garcia Viswanathan Natarajan 《The Journal of biological chemistry》2009,284(22):15339-15352
Phosphatidic acid generated by the activation of phospholipase D (PLD)
functions as a second messenger and plays a vital role in cell signaling. Here
we demonstrate that PLD-dependent generation of phosphatidic acid is critical
for Rac1/IQGAP1 signal transduction, translocation of
p47phox to cell periphery, and ROS production. Exposure of
[32P]orthophosphate-labeled human pulmonary artery endothelial
cells (HPAECs) to hyperoxia (95% O2 and 5% CO2) in the
presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as
evidenced by accumulation of [32P]phosphatidylbutanol. Infection of
HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated
hyperoxia-induced PLD activation and accumulation of
/reactive oxygen species
(ROS). Conversely, overexpression of catalytically inactive mutants of PLD
(hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1
or PLD2 siRNA did not augment hyperoxia-induced
[32P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia
caused rapid activation and redistribution of Rac1, and IQGAP1 to cell
periphery, and down-regulation of Rac1, and IQGAP1 attenuated
hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS
generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to
membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA,
suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally,
small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin
tyrosine phosphorylation and abolished Src, cortactin, and
p47phox redistribution to cell periphery. These results
demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1
in tyrosine phosphorylation of Src and cortactin, as well as in
p47phox translocation and ROS formation in human lung
endothelial cells.Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes,
and macrophages) generate superoxide
()2
instrumental in the killing of invading pathogens solely by NADPH oxidase
(1-3).
Deficiency of results in
the genetically inherited disorder chronic granulomatous disease, a condition
in which the affected individuals are susceptible to infection
(4). Phagocytic NADPH oxidase
is activated when cytosolic p47phox,
p67phox, and Rac2 translocate to the phagosomes and plasma
membrane and form a complex with integral membrane cytochrome
b558, which, in turn, is a Nox2
(gp91phox)/p22phox heterodimer
(5,
6). Assembly of phagocytic
NADPH oxidase is initiated by two signals. The first is the phosphorylation of
multiple serine and tyrosine residues in the p47phox
domain, which leads to unmasking of p47phox SH3 domains
that bind to a proline-rich target in the C terminus of
p22phox
(7-10).
The interaction between p47phox and
p22phox seems to be an essential requirement for the
translocation of other cytosolic components of the oxidase. The second signal
is the binding of GTP to Rac2, which leads to the dissociation of Rac from
Rho-GDI and binding to p67phox, followed by translocation
of p67phox/GTP-Rac2 to the membrane
(11). Nonphagocytic cells
express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5,
and most of the other cytosolic phagocytic oxidase components
(12); however, the oxidative
output of non-phagocytes is much smaller compared with the phagocytes. A
recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to
actin, thereby enhancing ROS production and contributing to cell motility in
ECs (13). The one or more
mechanisms responsible for differences in the oxidative burst between the
phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial
NADPH oxidase, which in part is mediated by ERK, p38 MAPK
(14,
15), and Src
(16), and hyperoxia-induced
p47phox tyrosine phosphorylation and translocation to cell
periphery is dependent on Src
(16). Further, tyrosine
phosphorylation of cortactin mediated by Src is essential for
hyperoxia-induced p47phox translocation and
/ROS generation in HPAECs
(17). In addition to Src,
phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of
p47phox and p22phox in neutrophils
both in vivo and in vitro
(18-20).
PA is generated in mammalian cells via de novo biosynthesis or
hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD)
(21-25).
Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced
the oxidative burst that correlated with PA accumulation, and inclusion of
short-chain primary alcohols attenuated the NADPH oxidase mediated
/ROS generation,
suggesting a potential role for PLD in the regulation of NADPH oxidase
(12,
26,
27). However, the downstream
targets of PLD that signal NADPH oxidase activation have not been fully
characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is
downstream of PLD in hyperoxia-induced ROS generation. In addition, we show
that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine
phosphorylation of cortactin and p47phox translocation to
cell periphery. Thus, our results define a novel molecular mechanism for
hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated
p47phox membrane translocation via
Rac1/IQGAP1/Src/cortactin signaling cascade. 相似文献
8.
Usatyuk PV Romer LH He D Parinandi NL Kleinberg ME Zhan S Jacobson JR Dudek SM Pendyala S Garcia JG Natarajan V 《The Journal of biological chemistry》2007,282(32):23284-23295
Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells. 相似文献
9.
Michael Hornsby Marcin Paduch Shane Miersch Annika S??f Tet Matsuguchi Brian Lee Karolina Wypisniak Allison Doak Daniel King Svitlana Usatyuk Kimberly Perry Vince Lu William Thomas Judy Luke Jay Goodman Robert J. Hoey Darson Lai Carly Griffin Zhijian Li Franco J. Vizeacoumar Debbie Dong Elliot Campbell Stephen Anderson Nan Zhong Susanne Gr?slund Shohei Koide Jason Moffat Sachdev Sidhu Anthony Kossiakoff James Wells 《Molecular & cellular proteomics : MCP》2015,14(10):2833-2847
10.
Dudek SM Camp SM Chiang ET Singleton PA Usatyuk PV Zhao Y Natarajan V Garcia JG 《Cellular signalling》2007,19(8):1754-1764
Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability. 相似文献