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An oncogenic form of the Flt-1 kinase has a tubulogenic potential in a sinusoidal endothelial cell line 总被引:2,自引:0,他引:2
We have previously reported a constitutively activated form of the Flt-1 kinase (BCR-FLTm) molecularly engineered based on the structural backbone of the activated tyrosine kinase BCR-ABL. Here we show that it can induce not only growth stimulation but also tubulogenic differentiation of non-tubulogenic NP31 (non parenchymal) sinusoidal endothelial cells of rat liver in basement membrane matrix. Tubules formed in vitro were accompanied by fenestration structures and allowed circulation when transplanted into syngeneic animals. This biological response was not observed in other activated forms of kinases constructed in a similar fashion, which include Trk (BCR-TRK), KDR (BCR-KDR), and the parental BCR-ABL. Interestingly, formation of fine tubules was accomplished with lower but not higher expression levels of BCR-FLTm. Compared to NP cells in primary culture NP31 is deficient in expression of alpha1 integrin subunit, which was restored by expression of BCR-FLTm that had tubulogenic ability. Matrix-induced tyrosine phosphorylation of an adaptor protein Shc with recruitment of Grb-2 was observed even when tubulogenesis was nearly completed at G1 stage of the cell cycle in 2-3 weeks. Activation of matrix metalloproteinase 2 (MMP-2) and expression of urokinase type plasminogen activator (uPA) was observed with cellular invasion into matrix at the depth of 200-300 microm. Inhibitors for MAP kinase activator MEK1 and for serine proteases showed deleterious effects on the tubulogenesis. We suppose that matrix ligand-induced integrin signals cooperate with a low level of Flt-1 kinase activity to promote tubulogenic behaviors of endothelial cells in this system. 相似文献
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Donny Hanjaya‐Putra Jane Yee Doug Ceci Rachel Truitt Derek Yee Sharon Gerecht 《Journal of cellular and molecular medicine》2010,14(10):2436-2447
Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co‐regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1‐MMP (MT1‐MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs. 相似文献
4.
We used two approaches to investigate the role of Src family kinases (SFKs) in ligand-stimulated internalization of the platelet-derived growth factor alpha receptor (alphaPDGFR). In cells that normally express SFKs, the internalization rate of the F72/74 mutant alphaPDGFR (which is unable to recruit or activate SFKs) was slower than that of the wild-type (WT) alphaPDGFR. When expressed in cells lacking SFKs (SYF cells), internalization of the WT and mutant receptors was indistinguishable, which indicated that there was not an inherent defect in the mutant receptor's ability to undergo ligand-driven internalization. The internalization difference between the WT and mutant receptors was seen again when c-Src was expressed in the SYF cells. Surprisingly, c-Src slowed the internalization of the mutant receptor but had little effect on WT receptor. We propose the following working hypothesis to explain these findings. In resting cells SFKs suppress internalization of the alphaPDGFR by phosphorylating a hypothetical protein X. This suppression is relieved when cells are exposed to PDGF because SFKs are recruited to the activated WT alphaPDGFR and thereby no longer actively phosphorylate protein X. The internalization of the mutant receptor is slow because it is unable to recruit SFKs and thereby fails to relieve the suppression of internalization. Our findings suggest a role for SFKs in regulating the permissiveness for internalization of the alphaPDGFR. 相似文献
5.
Aspenström P 《Experimental cell research》2004,298(2):485-498
The mammalian verprolin family consists of three family members: WIP, WIRE and CR16. WIRE was recently found to bind to WASP and N-WASP and to have roles in regulating actin dynamics downstream of the platelet-derived growth factor β-receptor. In the current study, the WASP-binding domain of WIRE was identified, with the core of the binding motif encompassing amino acid residues 408–412. A stretch of aromatic amino acid residues close to the core motif also participates in WASP binding. Amino acid substitutions in each of these motifs abrogated WASP binding, suggesting that both motifs are involved in the binding of WIRE to WASP. Interestingly, WIRE mutants unable to bind WASP were still able to induce a reorganisation of the actin filament system, indicating that WASP did not participate in the signalling pathway that link WIRE to actin dynamics. In cells ectopically expressing WIRE, the endocytosis of the platelet-derived growth factor β-receptor was drastically reduced. However, in contrast to the effect on the actin filament system, the WIRE-induced ablation of the receptor endocytosis required an intact WASP-binding domain. Moreover, WIRE was more efficient than WIP in inhibiting the receptor endocytosis, implicating that these two mammalian verprolins have distinct roles in mammalian cells. 相似文献
6.
Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 104 HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 104. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 104. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin. 相似文献
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Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor 总被引:13,自引:0,他引:13
Lang T Ikejima K Yoshikawa M Enomoto N Iijima K Kitamura T Takei Y Sato N 《Biochemical and biophysical research communications》2004,323(3):1091-1095
In the present study, we investigated the effect of leptin on proliferation of hepatic stellate cells (HSCs) in vitro. Proliferation of 3-day cultured rat HSCs was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the nuclei. The percentages of BrdU-positive cells were increased in the presence of PDGF-BB (5 ng/ml) for 8h as expected. Co-incubation with leptin (10-100 nM) potentiates this PDGF-dependent increase in BrdU positive cells in a dose-dependent manner. Messenger RNA for PDGF receptor alpha and beta subunits was increased almost 2- to 3-fold by incubation with leptin for 6h. Further, pre-incubation with leptin for 6h enhanced PDGF-induced increases in phospho-p44/42 MAP kinase and phospho-Akt levels in a dose-dependent manner. In the same condition, however, leptin per se did not increase phospho-STAT 3 and phospho-p44/42 MAP kinase levels. Instead, leptin increased phospho-Akt levels in HSCs within 30 min, suggesting that the phosphatidylinositol 3 kinase (PI3K)/Akt pathway is involved in the mechanism by which leptin accelerates the proliferation of HSCs. In conclusion, the present study clearly indicated that leptin potentiates PDGF-dependent proliferative responses of HSCs in vitro. 相似文献
9.
Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase
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Persson C Sävenhed C Bourdeau A Tremblay ML Markova B Böhmer FD Haj FG Neel BG Elson A Heldin CH Rönnstrand L Ostman A Hellberg C 《Molecular and cellular biology》2004,24(5):2190-2201
The platelet-derived growth factor (PDGF) beta receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF beta receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF beta receptor, we compared PDGF beta receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF beta receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cgamma1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cgamma1 activity and migratory hyperresponsiveness to PDGF. PDGF beta receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPepsilon ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF beta receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors. 相似文献
10.
The platelet-derived growth factor beta receptor as a target of the bovine papillomavirus E5 protein
The 44-amino acid E5 protein of bovine papillomavirus is a homo-dimeric, transmembrane protein that transforms cells by activating the platelet-derived growth factor ß receptor in a ligand-independent fashion. The E5 protein induces receptor activation by forming a stable complex with the receptor, thereby inducing receptor dimerization, trans-phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, and recruitment of cellular SH2 domain-containing proteins into a signal transduction complex. Direct interactions between specific transmembrane and juxtamembrane amino acids in the E5 protein and the PDGF ß receptor appear to drive complex formation and dimerization of the receptor. Further analysis of this unique mechanism of viral transformation promises to yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways. 相似文献
11.
Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors 总被引:2,自引:0,他引:2
There is now accumulating evidence that bone marrow-derived mesenchymal stem cells (MSCs) make an important contribution to postnatal vasculogenesis, especially during tissue ischaemia and tumour vascularization. Identifying mechanisms which regulate the role of MSCs in vasculogenesis is a key therapeutic objective, since while increased neovascularization can be advantageous during tissue ischaemia, it is deleterious during tumourigenesis. The potent angiogenic stimulant vascular endothelial growth factor (VEGF) is known to regulate MSC mobilization and recruitment to sites of neovascularization, as well as directing the differentiation of MSCs to a vascular cell fate. Despite the fact that MSCs did not express VEGF receptors, we have recently identified that VEGF-A can stimulate platelet-derived growth factor (PDGF) receptors, which regulates MSC migration and proliferation. This review focuses on the role of PDGF receptors in regulating the vascular cell fate of MSCs, with emphasis on the function of the novel VEGF-A/PDGF receptor signalling mechanism. 相似文献
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Mitra Esfandiarei Sahar Abdoli Yazdi Virginia Gray Shoukat Dedhar Cornelis van Breemen 《BMC cell biology》2010,11(1):16
Background
Vascular smooth muscle cell migration and accumulation in response to growth factors extensively contribute to the development of intimal thickening within the vessel wall. Cumulative evidence has shown that actin cytoskeleton polymerization and rearrangement are critical steps during cellular spreading and migration. Integrin-linked kinase, an intracellular serine/threonine kinase, is a cytoplasmic interactor of integrin beta-1 and beta-3 receptors regulating cell-cell and/or cell-extracellular matrix interaction, cell contraction, extracellular matrix modification, and cell spreading and migration in response to various stimuli. However, the regulatory role of ILK during vascular smooth muscle cell migration and the importance of integrin signaling in occlusive vascular diseases are not yet fully elucidated. 相似文献13.
Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer. 总被引:26,自引:8,他引:18
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Human platelet-derived growth factor (PDGF) occurs as three isoforms which are made up of disulfide-bonded A and B chains. The isoforms bind with different affinities to two different but structurally related cell surface receptors. The A type receptor binds all three isoforms (PDGF-AA, PDGF-AB, PDGF-BB) with high affinity, whereas the B type receptor binds PDGF-BB with high affinity, PDGF-AB with lower affinity but does not appear to bind PDGF-AA. We have utilized the differential effects of the three isoforms on actin reorganization and membrane ruffling in human foreskin fibroblasts to probe the idea that ligand-induced receptor dimerization is associated with receptor activation. Actin reorganization was found to be induced only by PDGF-AB and PDGF-BB and is therefore likely to be mediated by the B type receptor. Simultaneous addition of PDGF-AA, or downregulation of the A type receptor blocked the effect of PDGF-AB but not that of PDGF-BB. This is compatible with a model by which PDGF-AB binds to and dimerizes one A and one B type receptor; PDGF-AB therefore requires A type receptors in order to be functionally active at physiological concentrations. In cells with down-regulated A type receptors, high concentrations of PDGF-AB inhibited the effect of PDGF-BB on actin reorganization. We believe that this is due to a monovalent binding of PDGF-AB to the B type receptors which prevents PDGF-BB from dimerizing the receptors.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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The platelet-derived growth factor (PDGF) family consists of four isoforms which are secreted as homodimers (PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD) or heterodimers (PDGF-AB), and two receptor chains (PDGFR-α and -β). All members of the PDGF system are constitutively or inducibly expressed in renal cells and are involved in the regulation of cell proliferation and migration, the accumulation of extracellular matrix proteins and the secretion of pro- and anti-inflammatory mediators. Particular roles have been identified in mediating mesangioproliferative changes, renal interstitial fibrosis and glomerular angiogenesis. Different endogenous inhibitors of PDGF-induced biological responses exist which affect the activation/deactivation of PDGF isoforms, the activity of the PDGFRs, or which block downstream signaling pathways of the autophosphorylated PDGFRs. The novel endogenous inhibitor nephroblastoma overexpressed gene (NOV, CCN3) reduces PDGF-induced cell proliferation and is downregulated by PDGF isoforms itself. Among all identified inhibitors only few "true" PDGF antagonists have been identified. A better understanding of these inhibitors may aid in the design of novel therapeutic approaches to PDGF-mediated diseases. 相似文献
15.
A clonal human glioma cell line, U-343 MGa 31L, which expresses the A-type but not the B-type receptor for platelet-derived growth factor (PDGF), was used in a functional study of the A-type receptor. PDGF-AA induced, in a dose- and time-dependent manner, phosphorylation on tyrosine residues of the receptor in metabolically labelled cells. The optimal dose was around 30 ng/ml; at 100 ng/ml, phosphorylation was maximal at 15 min and had almost returned to the control level after 60 min. The phosphorylation on tyrosine residues of the PDGF A-type receptor was stimulated by PDGF-AA, PDGF-AB and PDGF-BB; these isoforms also stimulated [3H]thymidine incorporation into U-343 MGa 31L cells. In addition, activation of the A-type PDGF receptor induced transmodulation of the epidermal growth factor receptor. 相似文献
16.
D D Woo S P Fay R Griest W Coty I Goldfine C F Fox 《The Journal of biological chemistry》1986,261(1):460-467
Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites. 相似文献
17.
Activation of the protein kinase Akt/PKB mediates VEGF-dependent endothelial cell survival and eNOS activation. Here we examined the role of PKC in mediating VEGF-induced Akt activation. The PKC inhibitors GF109203X and calphostin C inhibited VEGF-induced Akt activation. Rottlerin and Go6976, inhibitors with specificities for PKC delta and alpha, respectively, also strongly inhibited VEGF-induced Akt activation. VEGF-induced Akt activation was prevented by down-regulation of PKC induced by prolonged pretreatment with the phorbol ester, PMA. VEGF induced phosphorylation of PKC delta at Thr 505 in the activation loop, and this phosphorylation was inhibited by LY294002, suggesting that modulation of PKC delta activation by VEGF occurs distal to phosphatidylinositol 3'-kinase. PKC and PI3K inhibitors both strongly reduced the stimulation of branching tubulogenesis by VEGF in vitro. The finding that PKC mediates VEGF-induced Akt activation identifies a novel signal transduction pathway through which Akt can be regulated by growth factors acting through receptor protein tyrosine kinases, and indicates that PKC-mediated Akt activity may play an essential role in VEGF-stimulated angiogenesis. 相似文献
18.
A role for platelet-derived growth factor in normal gliogenesis in the central nervous system 总被引:49,自引:0,他引:49
The bipotential progenitor cells (O-2A progenitors) that produce oligodendrocytes and type-2 astrocytes in the developing rat optic nerve are induced to proliferate in culture by type-1 astrocytes. Here, we show that the astrocyte-derived mitogen is platelet-derived growth factor (PDGF). PDGF is a potent mitogen for O-2A progenitor cells in vitro. Mitogenic activity in astrocyte-conditioned medium comigrates with PDGF on a size-exclusion column, competes with PDGF for receptors, and is neutralized by antibodies to PDGF. PDGF dimers can be immunoprecipitated from astrocyte-conditioned medium, and mRNA encoding PDGF is present in rat brain throughout gliogenesis. We propose that astrocyte-derived PDGF is crucial for the control of myelination in the developing central nervous system. 相似文献
19.
P L Orchansky J A Escobedo L T Williams 《The Journal of biological chemistry》1988,263(29):15159-15165
The platelet-derived growth factor (PDGF) receptor is usually anchored to the plasma membrane through a membrane-spanning hydrophobic amino acid sequence that splits the molecule into two approximately equal pieces, an amino-terminal external domain that contains the binding site for PDGF and a carboxyl-terminal cytoplasmic domain that includes the tyrosine kinase coding sequences. Here we report the expression of a truncated PDGF receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. Unexpectedly, this form of the receptor that lacks a hydrophobic membrane-anchoring sequence was bound to the membrane and was not secreted into the culture media. Conventional methods to dissociate noncovalent protein-protein interactions failed to release the protein from the membrane. When the transmembrane and cytoplasmic sequences were artificially deleted from the PDGF receptor, the truncated extracellular domain was anchored to the membrane through phospholipids and could be released by phospholipase C treatment. This truncated form of the receptor bound PDGF with an affinity 5-20-fold lower than the full-length receptor. 相似文献
20.
Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts 总被引:10,自引:0,他引:10
We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation. 相似文献