c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.     

Sphingosine 1-phosphate and platelet-derived growth factor (PDGF) act via PDGF beta receptor-sphingosine 1-phosphate receptor complexes in airway smooth muscle cells

Platelet-derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) act via PDGF beta receptor-S1P(1) receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S1P(1) antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF beta receptor-S1P(1) receptor complex. Third, treatment of cells with antisense S1P(1) receptor plasmid construct reduced the PDGF- and S1P-dependent activation of p42/p44 MAPK. Fourth, S1P and/or PDGF induced the formation of endocytic vesicles containing both PDGF beta receptors and S1P(1) receptors, which was required for activation of the p42/p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the presentation of sphingosine from other cell types and its conversion to S1P by the kinase exported from airway smooth muscle cells might enable S1P to act with PDGF on the PDGF beta receptor-S1P(1) receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-protein-coupled receptor agonists and/or sequential release and action of S1P in response to PDGF.     

Eicosapentaenoic acid inhibits PDGF-induced mitogenesis and cyclin D1 expression via TGF-beta in mesangial cells

Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid derived from fish oil, is efficacious in glomerular diseases where mesangial proliferation is a key event. We examined the mechanisms of action of EPA on platelet-derived growth factor (PDGF)-stimulated rat mesangial cell mitogenesis. EPA dose-dependently inhibited PDGF-stimulated [(3)H]-thymidine incorporation. PDGF-induced PDGF receptor autophosphorylation, an initial event for PDGF signaling, was not affected by 2 micro g/ml EPA. Similarly, PDGF-stimulated activation of extracellular signal-regulated kinase (ERK) was not altered. On the other hand, EPA inhibited cyclin-dependent kinase 4 (CDK4) activation and cyclin D1 protein induction, a critical step for G1/S progression. TGF-beta secretion assessed by ELISA and bioassay was increased by EPA at 18 h. Coincubation with anti-TGF-beta antibody inhibited the EPA-induced suppression of [(3)H]-thymidine incorporation and cyclin D1 expression. SB203580, an inhibitor of p38, a downstream kinase of TGF-beta, did not affect EPA's growth inhibitory effect. These results demonstrate that EPA inhibits PDGF-stimulated mesangial cell mitogenesis and cyclin D1 expression via TGF-beta.     

S1P stimulates chemotactic migration and invasion in OVCAR3 ovarian cancer cells

OVCAR3 ovarian cancer cells express three sphingosine 1-phosphate (S1P) receptors, S1P(1), S1P(2), and S1P(3), but not S1P(4). Stimulation of OVCAR3 cells with S1P induced intracellular calcium increases, which were partly inhibited by VPC 23019 (an S1P(1/3) antagonist). S1P-induced calcium increases were mediated by phospholipase C and pertussis toxin (PTX)-sensitive G-proteins in OVCAR3 cells. S1P stimulated extracellular signal-regulated kinase, p38 kinase, and Akt which were inhibited by PTX. S1P-stimulated chemotactic migration of OVCAR3 cells in a PTX-sensitive manner, indicating crucial role of G(i) protein(s) in the process. S1P-induced chemotactic migration of OVCAR3 cells was completely inhibited by LY294002 and SB203580. Pretreatment of VPC 23019 (an S1P(1/3) antagonist) completely inhibited S1P-induced chemotaxis. S1P also induced invasion of OVCAR3 cells, which was also inhibited by VPC 23019. Taken together, this study suggests that S1P stimulate chemotactic migration and cellular invasion, and VPC 23019-sensitive S1P receptor(s) might be involved in the processes.     

Sphingosine 1-phosphate, lysophosphatidic acid and growth factor signaling and termination

Sphingosine 1 phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lipid phosphates that bind to cell surface G-protein coupled receptors (GPCR) and, in addition, exhibit intracellular actions. We have summarised herein, an important functional interaction between lipid phosphate GPCR and receptor tyrosine kinases (RTK) that enables growth factors to spatially regulate effectors, thereby governing the nature of the biological response. For instance, we describe how the formation of functional complexes between the S1P(1) receptor and PDGFbeta receptor may effectively re-programme platelet-derived growth factor from a mitogenic to a migratory stimulus. This is achieved by integration of RTK- and GPCR-specific signals that results in spatial regulation of a cytoplasmic retained pool of extracellular signal regulated kinase-1/2 linked to myosin light chain kinase, myosin light chain phosphorylation and migration. We therefore suggest that the lipid phosphate receptor is a major determinant in regulating growth factor-dependent biology. Growth factors can also increase S1P inside cells, and we discuss the concept of spatial/temporal aspects of compartmentalised intracellular signaling of S1P in relation to defined interactions between, for instance, sphingosine kinase, phospholipase D1 and lipid phosphate phosphatases and regulation of cell survival.     

The effect of RGS12 on PDGFbeta receptor signalling to p42/p44 mitogen activated protein kinase in mammalian cells

We have previously shown that the PDGFbeta receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFbeta receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFbeta receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFbeta receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFbeta receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFbeta receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFbeta receptor in ASM cells.     

Phorbol ester downregulates PDGFbeta receptor via PKCbeta1 in vascular smooth muscle cells

The role of protein kinase C (PKC) and their isoforms in cell growth regulation remains elusive. Here we showed that in cultured human vascular smooth muscle cells (SMC), the PKC stimulator phorbol 12-myristate 13-acetate (PMA) inhibited [(3)H]thymidine incorporation in response to the growth factor PDGF associated with downregulation of PDGFbeta (but not alpha) receptors, which was recovered to normal level after PKC was depleted. The changes in PDGFbeta receptor were inversely correlated with PKCbeta1 protein levels regulated by PMA. The downregulation of PDGFbeta receptor by PMA was fully prevented by the PKCbeta inhibitor LY379196, however, without recovery of [(3)H]thymidine incorporation to PDGF. In contrast, [(3)H]thymidine incorporation was fully recovered after depletion of PKCs. These results indicate that in human SMC PKCbeta1 mediates PDGFbeta receptor downregulation. Other PKC isoforms activated by phorbol ester also contribute to the inhibitory effects on cell growth.     

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.     

Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P(2), S1P(3), S1P(4), but not S1P(1). When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P(1)- and S1P(4)-selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G(i) protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process.     

High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase

Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.     

S1P2 receptor mediates sphingosine-1-phosphate-induced fibronectin expression via MAPK signaling pathway in mesangial cells under high glucose condition

Accumulation of extracellular matrix including fibronectin in mesangium is one of the major pathologic characteristics in diabetic nephropathy. In the current study, we explored role of sphingosine-1-phosphate (S1P) receptor in fibronectin expression and underlying molecular mechanism. Among five S1P receptors the mRNA level of S1P2 receptor was the most abundant in kidney of diabetic rats and mesangial cells under high glucose condition. S1P augmentation of fibronectin was significantly inhibited by S1P2 receptor antagonist JTE-013 and S1P2-siRNA. S1P-stimulated fibronectin expression was remarkably blocked by ERK1/2 inhibitor PD98059 and p38MAPK inhibitor SB203580. Phospho-ERK1/2 and phospho-p38MAPK level induced by S1P were markedly abrogated by JTE-013 and S1P2-siRNA. In conclusion, S1P2 receptor was significantly up-regulated under diabetic condition. S1P2 receptor mediated fibronectin expression through the activation of S1P-S1P2-MAPK (ERK1/2 and p38MAPK) axis in mesangial cells under high glucose condition, suggesting that it might be a potential therapeutic target for diabetic nephropathy treatment.     

Sphingosine 1-phosphate induces cyclooxygenase-2 via Ca2+-dependent, but MAPK-independent mechanism in rat vascular smooth muscle cells

The effects of sphingosine 1-phosphate (S1P) on prostaglandin I(2) (PGI(2)) production and cyclooxygenase (COX) expression in cultured rat vascular smooth muscle cells (VSMCs) were investigated. S1P stimulated PGI(2) production in a concentration-dependent manner, which was completely suppressed by NS-398, a selective COX-2 inhibitor, as determined by radioimmunoassay. S1P stimulated COX-2 protein and mRNA expressions in a concentration- and time-dependent manner, while it had no effect on COX-1 expression. S1P(2) and S1P(3) receptors mRNA were abundantly expressed in rat VSMCs. Suramin, an antagonist of S1P(3) receptor, almost completely inhibited S1P-induced COX-2 expression. Pretreatment of VSMCs with pertussis toxin (PTX) partially, but significantly inhibited S1P-induced PGI(2) production and COX-2 expression. S1P also activated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). However, neither PD 98059, a selective inhibitor of ERK activation, nor SB 203580, a selective inhibitor of p38 MAPK, had a significant inhibitory effect on S1P-induced COX-2 expression, suggesting that the MAPK activation does not play main roles in S1P-induced COX-2 induction. S1P-induced COX-2 expression was inhibited by PP2, an inhibitor of Src-family tyrosine kinase, Ca(2+) depletion, and GF 109203X, an inhibitor of protein kinase C (PKC). These results suggest that S1P stimulates COX-2 induction in rat VSMCs through mechanisms involving Ca(2+)-dependent PKC and Src-family tyrosine kinase activation via S1P(3) receptor coupled to PTX-sensitive and -insensitive G proteins.     

Specific and overlapping sphingosine-1-phosphate receptor functions in human synoviocytes: impact of TNF-alpha

Sphingosine-1-phosphate (S1P), via interaction with its G protein-coupled receptors, regulates various physiological and pathological responses. The present study investigated the role of S1P/S1P receptor signaling in several functional responses of human fibroblast-like synoviocytes (FLSs) that may contribute to the pathogenesis of rheumatoid arthritis (RA). We report that FLSs express the S1P(1), S1P(2), and S1P(3) receptors. Moreover, exogenously applied S1P induces FLS 1) migration, 2) secretion of inflammatory cytokines/chemokines, and 3) protection from apoptosis. Using specific S1P receptor agonists/antagonists, we determined that S1P stimulates FLS migration through S1P(1) and S1P(3), induces cytokine/chemokine secretion through S1P(2) and S1P(3), and protects from cell apoptosis via S1P(1). The S1P-mediated cell motility and cytokine/chemokine secretion seem to be regulated by the p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and Rho kinase signal transduction pathways. Interestingly, treatment of FLSs with tumor necrosis factor-alpha increases S1P(3) expression and correlates with the enhancement of S1P-induced cytokine/chemokine production. Our data suggest that S1P(1), S1P(2), and S1P(3) play essential roles in the pathogenesis of RA by modulating FLS migration, cytokine/chemokine production, and cell survival. Moreover, the cytokine-rich environment of the inflamed synovium may synergize with S1P signaling to exacerbate the clinical manifestations of this autoimmune disease.     

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.     

Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells

Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24-48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation.     

Sphingosine-1-phosphate induces human endothelial VEGF and MMP-2 production via transcription factor ZNF580: Novel insights into angiogenesis

Sphingosine-1-phosphate (S1P)-induced migration and proliferation of endothelial cells are critical for angiogenesis. C2H2-zinc finger (ZNF) proteins usually play an essential role in altering gene expression and regulating the angiogenesis. The aim of this study is to investigate whether a novel human C2H2-zinc finger gene ZNF580 (Gene ID: 51157) is involved in the migration and proliferation of endothelial cells stimulated by S1P. Our study shows that EAhy926 endothelial cells express S1P1, S1P3 and S1P5 receptors. Furthermore, S1P upregulates both ZNF580 mRNA and protein levels in a concentration- and time-dependent manner. SB203580, the specific inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, blocks the S1P-induced upregulation of ZNF580. Moreover, overexpression/downexpression of ZNF580 in EAhy926 cells leads to the enhancement/decrease of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) expression as well as the migration and proliferation of EAhy926 endothelial cells. These results elucidate the important role that ZNF580 plays in the process of migration and proliferation of endothelial cells, which provides a foundation for a novel approach to regulate angiogenesis.     

Platelet-derived growth factor-stimulated migration of murine fibroblasts is associated with epidermal growth factor receptor expression and tyrosine phosphorylation

Previous studies have shown that epidermal growth factor (EGF) synergizes with various extracellular matrix components in promoting the migration of B82L fibroblasts expressing wild-type EGF receptors and that functional EGF receptors are critical for the conversion of B82L fibroblasts to a migratory cell type (). In the present study, we examined the effects of platelet-derived growth factor (PDGF) on the motility of B82L fibroblasts using a microchemotaxis chamber. We found that PDGF can enhance fibronectin-induced migration of B82L fibroblasts expressing wild-type EGF receptors (B82L-clone B3). However, B82L cells that lack the EGF receptor (B82L-parental) or that express an EGF receptor that is kinase-inactive (B82L-K721M) or C-terminally truncated (B82L-c'973) exhibit little PDGF-stimulated migration. In addition, none of these three cell lines exhibit the capacity to migrate to fibronectin alone. These observations indicate that, similar to cell migration toward fibronectin, PDGF-induced cell migration of B82L fibroblasts is augmented by the expression of an intact EGF receptor kinase. The loss of PDGF-stimulated motility in B82L cells that do not express an intact EGF receptor does not appear to result from a gross dysfunction of PDGF receptors, because ligand-stimulated tyrosine phosphorylation of the PDGF-beta receptor and the activation of mitogen-activated protein kinases are readily detectable in these cells. Moreover, an interaction between EGF and PDGF receptor systems is supported by the observation that the EGF receptor exhibits an increase in phosphotyrosine content in a time-dependent fashion upon the addition of PDGF. Altogether, these studies demonstrate that the expression of EGF receptor is critical for PDGF-stimulated migration of murine B82L fibroblasts and suggest a role for the EGF receptor downstream of PDGF receptor activation in the signaling events that lead to PDGF-stimulated cell motility.