Akt-mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.     

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.     

p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity

We show in rat lung microvessel endothelial cells (RLMVEC) that endocytosis is a critical determinant of activation of mitogen-activated protein kinase (MAPK) and thereby regulates endothelial monolayer integrity. In RLMVEC grown in serum-free medium, we observed that albumin supplementation induced the phosphorylation of p38 MAPK within 30 min, which persisted for up to 2 h. Engagement of the endocytic machinery regulated the activation of p38 MAPK that contributed to endothelial cell proliferation and reduction of apoptosis. We also observed an interaction between the caveolar protein caveolin-1 and p38 MAPK with reciprocal coimmunoprecipitation assays and colocalization using double-label immunofluorescence staining. Knockdown of caveolin-1 expression with small interfering RNA significantly reduced endocytosis and activation of p38 MAPK and interfered with the ability of endothelial cells to form a confluent monolayer. Thus caveolae-mediated endocytosis and concomitant activation of p38 MAPK may help to maintain endothelial monolayer integrity by signaling proliferation and survival of endothelial cells.     

SIV-induced activation of the blood-brain barrier requires cell-associated virus and is not restricted to endothelial cell activation

It has never been determined if activation of the blood-brain barrier (BBB) during simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection is a function of high levels of circulating virus or if the virus has to be within a cell capable of crossing the BBB to activate it. In vitro models of the BBB are becoming recognized as an acceptable method for determining the cellular events associated with HIV neuroinvasion. Cell free virus (when added in the physiologically relevant lumen) although capable of activating the endothelial cells of our in vitro BBB did not activate astrocytes beneath. SIVmac251-infected CEMx174 cells, however, were capable of activating both components of the BBB model. Here we demonstrate that an in vitro model of the BBB can be activated in a physiologically relevant manner, that SIV requires to be cell-associated and that endothelial cells of the BBB are not the only components that are activated during SIV neuroinvasion.     

Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation

We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.     

Phospholipase cbeta is critical for T cell chemotaxis

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.     

Regulation of muscle satellite cell activation and chemotaxis by angiotensin II

The role of angiotensin II (Ang II) in skeletal muscle is poorly understood. We report that pharmacological inhibition of Ang II signaling or ablation of the AT1a receptor significantly impaired skeletal muscle growth following myotrauma, in vivo, likely due to impaired satellite cell activation and chemotaxis. In vitro experiments demonstrated that Ang II treatment activated quiescent myoblasts as evidenced by the upregulation of myogenic regulatory factors, increased number of β-gal+, Myf5-LacZ myoblasts and the acquisition of cellular motility. Furthermore, exogenous treatment with Ang II significantly increased the chemotactic capacity of C2C12 and primary cells while AT1a(-/-) myoblasts demonstrated a severe impairment in basal migration and were not responsive to Ang II treatment. Additionally, Ang II interacted with myoblasts in a paracrine-mediated fashion as 4 h of cyclic mechanical stimulation resulted in Ang II-induced migration of cocultured myoblasts. Ang II-induced chemotaxis appeared to be regulated by multiple mechanisms including reorganization of the actin cytoskeleton and augmentation of MMP2 activity. Collectively, these results highlight a novel role for Ang II and ACE inhibitors in the regulation of skeletal muscle growth and satellite cell function.     

Plasminogen N-terminal activation peptide modulates the activity of angiostatin-related peptides on endothelial cell proliferation and migration

Angiostatin, a potent inhibitor of angiogenesis, is derived from the fibrinolytic proenzyme, plasminogen, by enzymatic processing. Plasminogen N-terminal activation peptide (PAP) is one of the products concomitantly released aside from angiostatin (kringles 1-4) and mini-plasminogen (kringle 5 plus the catalytic domain) when plasminogen is processed. To determine whether PAP alone or together with the angiostatin-related peptides derived from the processing of plasminogen modulate the proliferation and motility of endothelial cells, we have generated a recombinant PAP and used it to study its effects on endothelial cells in the presence and absence of the angiostatin-related peptides. Our results showed that PAP alone slightly increased the migration but not the proliferation of endothelial cells. However, in the presence of the angiostatin-related peptides, PAP attenuated the inhibitory activity of the angiostatin-related peptides on the proliferation and migration of endothelial cells. The inhibitory effect of PAP on the angiostatin-related peptides could be due to its binding to the kringle domains of the latter peptides.     

Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen

Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.     

人源性激肽释放酶结合蛋白与内皮细胞功能

人源性激肽释放酶结合蛋白（Kallistatin,Kal）是一种负性急性期内源性蛋白,与多种内皮相关性生理和病理过程密切相关,如血管生成及损伤修复、炎症、心功能不全、肾损伤、糖尿病等。炎症和氧化应激可引起内皮功能障碍,而Kal可抑制肿瘤坏死因子α引起的内皮细胞活化,通过KLF4-eNOS、PI3K-AKT-eNOS和AKT-FOXO1等信号通路,增加内皮细胞NO合酶的表达和NO生成,抑制内皮细胞损伤和凋亡。动物实验显示,Kal表达增加可减弱氧化应激诱导的细胞凋亡和器官损伤。基于内皮细胞所处的状态或来源,如健康或损伤情况,成熟内皮细胞或内皮祖细胞,Kal的作用可能有所区别。内皮细胞是参与肿瘤生长与转移的关键因素已达成共识,但肿瘤新生血管形成的机制尚待确认。Kal可诱导肿瘤内皮细胞凋亡,抑制肿瘤新生血管生成和肿瘤生长的能力已被证实。临床前研究结果表明,Kal具有多种药理作用,对氧化应激相关性疾病,特别是肿瘤治疗具有应用前景,但其药理作用的分子机制仍需深入探讨。     

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, “classical chemoattractants” such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and “pure chemoattractants” such as TGF-β1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the “non-classical” group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca²⁺]_i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca²⁺-independent G-protein-coupled pathway for post-receptor signal transduction triggered by “pure chemoattractants.” The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds. © 1996 Wiley-Liss, Inc.     

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.     

Tumor-induced endothelial cell activation: role of vascular endothelial growth factor

Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl₂ significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl₂, both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects. vascular endothelial growth factor receptor 2; MG63-conditioned medium     

Analysis of the roles of microvessel endothelial cell random motility and chemotaxis in angiogenesis.

The growth of new capillary blood vessels, or angiogenesis, is a prominent component of numerous physiological and pathological conditions. An understanding of the co-ordination of underlying cellular behaviors would be helpful for therapeutic manipulation of the process. A probabilistic mathematical model of angiogenesis is developed based upon specific microvessel endothelial cell (MEC) functions involved in vessel growth. The model focuses on the roles of MEC random motility and chemotaxis, to test the hypothesis that these MEC behaviors are of critical importance in determining capillary growth rate and network structure. Model predictions are computer simulations of microvessel networks, from which questions of interest are examined both qualitatively and quantitatively. Results indicate that a moderate MEC chemotactic response toward an angiogenic stimulus, similar to that measured in vitro in response to acidic fibroblast growth factor, is necessary to provide directed vascular network growth. Persistent random motility alone, with initial budding biased toward the stimulus, does not adequately provide directed network growth. A significant degree of randomness in cell migration direction, however, is required for vessel anastomosis and capillary loop formation, as simulations with an overly strong chemotactic response produce network structures largely absent of these features. The predicted vessel extension rate and network structure in the simulations are quantitatively consistent with experimental observations of angiogenesis in vivo. This suggests that the rate of vessel outgrowth is primarily determined by MEC migration rate, and consequently that quantitative in vitro migration assays might be useful tools for the prescreening of possible angiogenesis activators and inhibitors. Finally, reduction of MEC speed results in substantial inhibition of simulated angiogenesis. Together, these results predict that both random motility and chemotaxis are MEC functions critically involved in determining the rate and morphology of new microvessel network growth.     

Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity.

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down-regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21(WAF/CIP)-dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21(WAF/CIP) nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression.     

Signal transduction pathways in mast cell granule-mediated endothelial cell activation

BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated protein kinase activation and nuclear factor-kappaB translocation.     

Conversion of Glu-plasminogen to Lys-plasminogen is necessary for optimal stimulation of plasminogen activation on the endothelial cell surface

When Glu-plasminogen is bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators is markedly enhanced compared with the reaction in solution. It is not known whether the direct activation of Glu-Pg by Pg activators is promoted on the cell surface or whether plasminolytic conversion of Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm formation on the cell surface. To distinguish between these potential mechanisms, we tested whether Pm formation on the cell surface could be stimulated in the absence of conversion of Glu-Pg to Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase (u-PA) were compared when these Pg forms were either bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg(561)-Val(562) bond by Pg activators but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp(646) of the serine protease catalytic triad.) Glu-Pg activation by t-PA was enhanced on HUVEC compared with the solution phase by 13-fold. In contrast, much less enhancement of Pg activation was observed with [D646E]Glu-Pg ( approximately 2-fold). Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when u-PA was used as activator. When Glu-Pg was bound to the cell in the presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected, consistent with the conversion of Glu-Pg to Lys-Pg being necessary for optimal enhancement of Pg activation on cell surfaces. Furthermore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was markedly enhanced ( approximately 20-fold) on the HUVEC surface, suggesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.