Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.     

Angiogenin activates human umbilical artery smooth muscle cells

Angiogenin stimulates proliferation of human umbilical artery smooth muscle cells. This activity of angiogenin depends on the cell density and requires nuclear translocation of the ligand as well as activation of SAPK/JNK MAP kinase. Angiogenin binds to a 170-kDa putative receptor on the cell surface and induces phosphorylation of SAPK/JNK. It also undergoes nuclear translocation in a time and concentration dependent manner. Neomycin inhibits nuclear translocation of angiogenin and abolishes angiogenin-induced cell proliferation but does not inhibit SAPK/JNK phosphorylation. The data demonstrate that smooth muscle cells are targets for angiogenin and that both SAPK/JNK phosphorylation and nuclear translocation of the ligand are required for angiogenin to activate smooth muscle cells.     

VEGF induces proliferation, migration, and TGF-beta1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-beta1 mRNA, enhanced TGF-beta1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-beta1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.     

Identification and characterization of follistatin as a novel angiogenin-binding protein

Angiogenin enhances tumorigenesis. However, the mechanisms of angiogenin-induced angiogenesis and cancer cell proliferation remain elusive. In this study, follistatin was identified as a binding partner of angiogenin by a yeast two-hybrid screen and confirmed by a pull-down experiment. The interaction of fluorescently tagged angiogenin and follistatin was monitored in real time by a laser confocal microscope and shown to localize at the sub-nuclear region of HeLa cells. Additional yeast two-hybrid analysis revealed that domains 2 and 3 of follistatin were the minimal structure requirement for angiogenin binding. These findings provide new clues for further studies on the mechanisms of angiogenin-induced angiogenesis or cancer cell growth.     

Examining the mechanism of Erk nuclear translocation using green fluorescent protein

In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2.     

Fluvastatin attenuates IGF-1-induced ERK1/2 activation and cell proliferation by mevalonic acid depletion in human mesangial cells

AimsInsulin-like growth factor (IGF)-1 is a major mitogenic growth factor for mesangial cells (MCs). Statins slow the progression of chronic kidney disease by affecting inflammatory cell signaling pathways, in addition to improving lipid profile, however, no studies have investigated the effects of fluvastatin on mitogen-activated protein (MAP) kinase activity or MC proliferation in kidney cells. We investigated the effects of fluvastatin on IGF-1-induced activation of intracellular signal pathways and MC proliferation, and examined the inhibitory mechanisms of fluvastatin.Main methodsWestern blotting and cell proliferation assay were used.Key findingsIGF-1 induced phosphorylation of extracellular-related kinase (ERK)1/2, MAP or ERK kinase (MEK)1/2, and Akt, expression of cyclin D1, and MC proliferation in cultured human MCs. Fluvastatin or PD98059, an MEK1 inhibitor, completely abolished IGF-1-induced MEK1/2 and ERK1/2 phosphorylation and MC proliferation, whereas inhibition of Akt had no effect on MC proliferation. Mevalonic acid prevented fluvastatin inhibition of IGF-1-induced MEK1/2 and ERK1/2 phosphorylation, cyclin D1 expression, and MC proliferation.SignificanceFluvastatin inhibits IGF-1-induced activation of the MAP kinase pathway and MC proliferation by mevalonic acid depletion, and might have renoprotective effects by inhibiting IGF-1-mediated MC proliferation.     

Activation of multiple signaling pathways is critical for fibroblast growth factor 2- and vascular endothelial growth factor-stimulated ovine fetoplacental endothelial cell proliferation

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by N(G)-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.     

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor.Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.     

Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2

The signaling mechanisms responsible for bone morphogenetic protein (BMP) induced osteoblast differentiation remains poorly understood. Previous research demonstrated that Smad proteins are the substrates and the mediators of BMP bound serine/threonine receptor kinase. In the present study, we examined the possible involvement of extracellular signal-regulated kinase (Erk) in the BMP induced osteoblast differentiation of mesenchymal progenitor cell C3H10T1/2. Our results indicate that BMP-2 inducement increased MAP kinase activity in mesenchymal progenitor cell line C3H10T1/2. Contrary to previous reports, this increased MAP kinase activity showed a latent but sustained pattern. Elevation of Erk1 and Erk2 protein levels was observed simultaneously. RT-PCR results demonstrated that the elevation of Erk protein level in BMP-2 induced cells was from the upregulation of mRNA expression. Furthermore, upregulated Erk proteins present enhanced phosphorylation. By using a dominant-negative Erk2 cell line, we demonstrated that nonfunctional Erk2 partially eliminated BMP-2 induced cell proliferation and ALP activity in the C3H10T1/2 cell. These results indicate that Erk is involved in BMP-2 induced osteoblast differentiation. The results also demonstrate that a latent and sustained signaling pattern exists in BMP induced signaling cascade.     

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.     

Limited Proteolysis of Angiogenin by Elastase Is Regulated by Plasminogen

Human neutrophil elastase cleaves angiogenin at the Ile-29/Met-30 peptide bond to produce two major disulfide-linked fragments with apparent molecular weights of 10,000 and 4000, respectively. Elastase-cleaved angiogenin has slightly increased ribonucleolytic activity, but has lost its ability to undergo nuclear translocation in endothelial cells, a process essential for angiogenic activity. Cleavage appears to alter the cell-binding properties of angiogenin, despite the fact that it occurs some distance from the putative receptor-binding site, since the elastase-cleaved protein fails to compete with its native counterpart for nuclear translocation in endothelial cells. Plasminogen specifically accelerates elastase proteolysis of angiogenin. It does not enhance elastase activity toward ribonuclease A or the synthetic peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Plasminogen-accelerated inactivation of angiogenin by elastase might be a significant event in the process of angiogenin-induced angiogenesis since (i) angiogenin and plasminogen circulate in plasma at high concentrations, (ii) angiogenin, especially when bound to actin, activates tissue plasminogen activator to generate plasmin from plasminogen, and (iii) elastase cleaves plasminogen to produce angiostatin, a potent inhibitor of angiogenesis and metastasis. Interrelationships among angiogenin, plasminogen, plasminogen activators, elastase, and angiostatin may provide a sensitive regulatory system to balance angiogenesis and antiangiogenesis.     

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenin’s propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.     

Chemokine receptors CXCR-1/2 activate mitogen-activated protein kinase via the epidermal growth factor receptor in ovarian cancer cells

Ovarian cancer typically disseminates widely in the abdomen, a characteristic that limits curative therapy. The mechanisms that promote ovarian cancer cell migration are incompletely understood. We studied model SK-OV-3 ovarian cancer cells and observed robust expression of the alpha chemokine receptors CXCR-1 and CXCR-2. Interleukin-8 (IL-8) treatment caused shape changes in the cells, with membrane ruffling and formation/retraction of thin actin-like projections, as detected by time-lapse microscopy. Stimulation of the CXCR-1/2 receptors by human interleukin 8 (IL-8) rapidly activated the p44/42 mitogen-activated protein (extracellular signal-regulated kinase (Erk1/2)) kinase pathway. Treatment of SK-OV-3 cells with the inhibitors genestein and herbimycin A indicated that tyrosine kinases were involved in the IL-8 activation of Erk1 and Erk2. Of note, IL-8 induced transient phosphorylation of the epidermal growth factor (EGF) receptor and its association with the adaptor molecules Shc and Grb2. This transactivation of the EGF receptor was dependent on intracellular Ca(2+) mobilization. Furthermore AG1478, a specific inhibitor of the EGF receptor kinase, blocked Erk1 and Erk2 activation. c-Src kinase was not involved in the IL-8-mediated phosphorylation of the EGF receptor, but was critical for Shc phosphorylation and downstream Erk1/2 kinase activation. These results suggest important "cross-talk" between chemokine and growth factor pathways that may link signals of cell migration and proliferation in ovarian cancer.     

Heparin suppresses lipid raft-mediated signaling and ligand-independent EGF receptor activation

Heparin is well known to suppress vascular smooth muscle cell (VSMC) proliferation, and attempts to exploit this therapeutically have led to recognition of multiple pathways for heparin's anti-mitogenic actions. At low concentrations (ca. 1 microg.ml(-1)), these suppressive effects may reflect physiological activities of endogenous heparan sulfates, and appear to be rapid responses to extracellular or cell surface-associated heparin. Because heparin has been shown to influence expression of caveolin proteins, and caveolae/lipid rafts are critical structures modulating cell signaling, we examined the effect of heparin on signaling involving cholesterol-rich membrane microdomains. The VSMC line PAC-1 activates the MAP kinase Erk in response to the cholesterol-sequestering agents methyl-beta-cyclodextrin and nystatin. This follows a temporal sequence that involves Ras-GTP activation of MEK, and is independent of PKC, Src, and PI3 kinase. However, ligand-independent phosphorylation of the EGF receptor (EGFR) by removal of cholesterol precedes Ras activation, and the EGFR kinase inhibitor AG1478 blocks Erk phosphorylation, supporting occurrence of the signaling sequence EGFR-Ras-MEK-Erk. Phosphorylation of EGFR occurs predominantly in caveolin-rich microdomains as identified by Western blotting of fractions from density gradient centrifugation of membranes prepared under detergent-free conditions. In these situations, heparin inhibits phosphorylation of EGFR on the Src-dependent site Tyr(845), but not the autophosphorylation of Tyr(1173), and decreases Ras activation and Erk phosphorylation. We conclude that heparin can suppress Erk signaling in VSMC with effects on site-specific phosphorylation of EGFR localized in caveolin-enriched lipid rafts.