Biomedical subjects

L Claesson-Welsh

Publications and source records attributed to L Claesson-Welsh.

At least 55 records · Page 3Linked to original sources

PubMed1998

PDGF alpha-receptor mediated cellular responses are not dependent on Src family kinases in endothelial cells.

Two novel autophosphorylation sites in the juxtamembrane region of the PDGF alpha-receptor, Tyr-572 and Tyr-574, were identified. A Y572/574F mutant PDGF (alpha)-receptor was generated and stably expressed in porcine aortic endothelial cells. In contrast to the wild-type receptor, the mutant receptor was unable to associate with or activate Src family tyrosine kinases. Tyrosine phosphorylated synthetic peptides representing the juxtamembrane sequence of the receptor dose-dependently inhibited the binding of Src family tyrosine kinases to the autophosphorylated PDGF alpha-receptor. The mutant receptor showed similar PDGF-induced kinase activity and ability to mediate mitogenicity, actin reorganization and chemotaxis as the wild-type receptor. Thus activation of Src family kinases by the PDGF alpha-receptor is not essential for PDGF-induced mitogenicity or actin reorganization.

Actins↗

PubMed1997

Hyaluronan oligosaccharides induce tube formation of a brain endothelial cell line in vitro.

In remodeling tissues the formation of new blood vessels is an essential process which is regulated by different factors. During such processes an increase in hyaluronan synthesis and turnover is often seen and recent observations have suggested that hyaluronan can both promote and inhibit neovascularization depending on its molecular mass. In this work we show that a brain capillary endothelial cell line forms tubes in a collagen gel after stimulation by hyaluronan oligosaccharides. Ultrastructural examination of the tubes by transmission electron microscopy revealed that the cord-like outgrowths consisted of 2-10 tightly packed cells containing a continuous lumen. The tube formation in response to hyaluronan oligosaccharide was not mediated by activation of receptors for fibroblast growth factor. The endothelial cell line, which does not synthesize hyaluronan, exhibited specific hyaluronan-binding sites, with about 3000 hyaluronan molecules (M(r) 3.85 x 10(6)) bound per cell at saturation and Kd was 0.05 x 10(-9) M. Furthermore, the cell line was stained with mAb IVd4 that recognizes a novel class of hyaluronan-binding proteins and mAb IM7 which recognizes CD44 molecules.

Animals↗

PubMed1997

Phosphatidylinositol 3'-kinase-independent p70 S6 kinase activation by fibroblast growth factor receptor-1 is important for proliferation but not differentiation of endothelial cells.

p70(s6k) has a role in cell cycle progression in response to specific extracellular stimuli. The signal transduction pathway leading to activation of p70(s6k) by fibroblast growth factor receptor-1 (FGFR-1) was examined in FGF-2-treated rat L6 myoblasts. p70(s6k) was activated in a biphasic and rapamycin-sensitive manner. Although phosphatidylinositol 3'-kinase was not activated in the FGF-2 treated cells, as judged from in vitro and in vivo analyses, wortmannin and LY294002 treatment inhibited p70(s6k) activation. Inhibition of protein kinase C (PKC), by bisindolylmaleimide or by chronic phorbol ester treatment of the FGFR-1 cells, suppressed but did not block p70(s6k) activation. In cells expressing a point-mutated FGFR-1, Y766F, unable to mediate PKC activation, p70(s6k) was still activated, in a bisindolylmaleimide- and phorbol ester-resistant manner. The involvement of S6 kinase in FGFR-1-dependent biological responses was examined in murine brain endothelial cells. In response to FGF-2, these cells differentiate to form tube-like structures in collagen gel cultures and proliferate when cultured on fibronectin. p70(s6k) was not activated in endothelial cells on collagen, whereas activation was observed during proliferation on fibronectin. In agreement with this finding, rapamycin inhibited the proliferative but not the differentiation response. Our results indicate that FGFR-1 mediates p70(s6k) activation by a phosphatidylinositol 3'-kinase-independent mechanism that does not require PKC activation and, furthermore, proliferation, but not differentiation of endothelial cells in response to FGF-2, is associated with p70(s6k) activation.

Animals↗

PubMed1997

Involvement of phosphatidylinositide 3'-kinase and Rac in platelet-derived growth factor-induced actin reorganization and chemotaxis.

Previous work has suggested a role for phosphatidylinositide 3'-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF beta-receptor expressing porcine aortic endothelial (PAE/PDGFR-beta) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC50 value of about 15-20 nM. Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-beta cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-beta cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-beta cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.

Actins↗

PubMed1997

Proteolytic processing regulates receptor specificity and activity of VEGF-C.

The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.

Amino Acid Sequence↗

PubMed1997

Phosphorylation of a 72-kDa protein in PDGF-stimulated cells which forms complex with c-Crk, c-Fyn and Eps15.

Ligand-induced activation of the beta-receptor for platelet-derived growth factor (PDGF) induces tyrosine phosphorylation of a number of downstream signaling proteins. In the present study, we used two-dimensional gel electrophoresis to characterize the spectrum of proteins phosphorylated in response to PDGF stimulation in porcine aortic endothelial cells expressing PDGF beta-receptors. Several previously known substrates for the PDGF beta-receptor were identified as well as a novel substrate of 72 kDa. The 72-kDa component could be co-immunoprecipitated in complex with the adaptor protein c-Crk, the non-receptor tyrosine kinase c-Fyn and the signaling molecule Eps15. The results obtained suggests that the 72-kDa protein might play an important role in signaling via the PDGF beta-receptor, coupling non-receptor tyrosine kinases of the Src family with c-Crk and Eps15.

Animals↗

PubMed1997

[Molecular mechanism in cell migration].

Lena Claesson-Welsh was awarded the Anders Jahre Prize for young scientists in 1996 in recognition of her research into growth factors and their regulation, knowledge of which is important for an understanding of cancer and angiogenesis. The ability of cells to move along a chemical concentration gradient (chemotaxis) is an important property in many physiological and pathological processes, such as embryogenesis, wound healing and angiogenesis. In angiogenesis, whereby new microcapillaries are formed as sprouts from existing vessels, vascular endothelial cells undergo a complex three-step process. The first step consists in focal degeneration mediated by proteases produced by growth factor-stimulated endothelial cells. The next step is concomitant proliferation and migration of cells into the surrounding tissue. Finally, the cells differentiate to form a continuous lumen. Tumour growth is strictly dependent on angiogenesis. Many tumours produce the growth factor. VEGF, which exerts its effect on receptor tyrosine kineases which are expressed on endothelial cells, thus stimulating endothelial cell chemotaxis toward the tumour. The regulation of endothelial cell migration in this pathological process would be of great therapeutic value.

Angiogenesis Inducing Agents↗

PubMed1996

Grb7 is a downstream signaling component of platelet-derived growth factor alpha- and beta-receptors.

Ligand stimulation of the platelet-derived growth factor (PDGF) alpha- or beta-receptors leads to activation of their intrinsic tyrosine kinases and autophosphorylation of tyrosine residues. Grb7 is an SH2 and PH domain-containing molecule that is known to be overexpressed in some breast cancer tissues and cell lines. Here we show that the SH2 domain of Grb7 can directly bind to the autophosphorylated PDGF beta-receptor in vitro. Grb7 association to the PDGF beta-receptor was dramatically reduced by replacement of tyrosine residues 716 or 775 with phenylalanine residues. Synthetic phosphorylated peptides containing Tyr-716 or Tyr-775 inhibited binding of the Grb7 SH2 domain to the autophosphorylated PDGF beta-receptor in a manner similar to but distinct from the binding of the Grb2 SH2 domain. Grb7 associated with activated PDGF beta-receptors in vivo, and the association was dramatically reduced by substitution of Tyr-716 or Tyr-775 with a phenylalanine residue. Furthermore, complex formation between Shc and Grb7 was observed after ligand stimulation of PDGF alpha- or beta-receptors in cells transfected with Grb7 cDNA or in the breast cancer cell line BT-474. Thus, Grb7 is implicated in PDGF signaling pathways in certain cell types by binding to the receptor directly or indirectly via Shc.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2.

In Swiss 3T3 fibroblasts, basic fibroblast growth factor (bFGF) stimulates the unique tyrosine phosphorylation of a protein complex around 90 kDa, as ascertained by high resolution 2-D PAGE and anti-phosphotyrosine blotting. The majority of this complex consists of the protein(s) designated previously as SNT (suc1-associated neurotrophic factor target). Tyrosine phosphorylated SNT binds to both p13suc1 protein and the SH2 domain of Grb2. Binding of SNT to Grb2 is likely to be mediated through the consensus binding motif, pYXN, on SNT. The binding of SNT to p13suc1 is independent of the pYXN motif. Tyrosine phosphorylated SNT is localised in the plasma membrane where it could form a complex with Grb2 and Sos, enabling the initiation of a novel FGF-specific signalling pathway.

3T3 Cells↗

PubMed1996

Stimulation of actin stress fibre formation mediated by activation of phospholipase D.

BACKGROUND: Agonist-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine, generating the putative messenger phosphatidate (PA). Proposed functions for PA, and hence for PLD, include kinase activation, the regulation of small molecular weight GTP-binding proteins, actin polymerization and secretion. It has not been possible to define a physiological function for PLD activation as it is generally stimulated together with other signalling pathways, such as those involving phospholipases A2 and C, phosphatidylinositide (PI) 3-kinase and the p21(ras)/mitogen-activated protein (MAP) kinase cascade. RESULTS: We report that, in porcine aortic endothelial (PAE) cells, lysophosphatidic acid (LPA) stimulated PLD activity and rapidly generated PA in the absence of other phospholipase, PI 3-kinase or MAP kinase activities. PLD activation was controlled by a tyrosine kinase-regulated pathway. LPA also stimulated actin stress fibre formation, but was inhibited by butan-1-ol; the alcohol also reduced the accumulation of PA. The addition of PA to cells did not stimulate PLD activity, but did cause stress fibre formation in a manner that was insensitive to butan-1-ol. Stimulation of stress fibre formation by LPA and PA was sensitive to genistein, and was inhibited by micro-injection of the Rho-inhibiting C3 exotoxin into PAE cells. CONCLUSIONS: This study provides the first clear demonstration of a physiological role for PLD activity. In PAE cells, the stimulation of actin stress fibre formation was a consequence of PA generation and, therefore, PLD activation. The results suggest that PA generation is upstream of Rho activation, and imply a role for PLD in the regulation of Rho-mediated pathways.

Actins↗

PubMed1996

Requirement for phosphatidylinositol 3'-kinase activity in platelet-derived growth factor-stimulated tyrosine phosphorylation of p125 focal adhesion kinase and paxillin.

The role of phosphatidylinositol 3'-kinase (PI 3'-kinase) activity in platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin has been examined. The tyrosine phosphorylation of p125FAK and paxillin in response to PDGF was markedly inhibited by wortmannin in a dose-dependent manner. PDGF-stimulated PI 3'-kinase activity, membrane ruffle formation, and tyrosine phosphorylation of p125FAK and paxillin were all inhibited by the same low concentrations of wortmannin (>90% inhibition at 40nM). In contrast, tyrosine phosphorylation of p125FAK and paxillin in response to bombesin, endothelin, and phorbol 12,13-dibutyrate was not inhibited by wortmannin in these cells. Furthermore, LY294002, an inhibitor of PI 3'-kinase structurally unrelated to wortmannin, also inhibited PDGF-stimulated p125FAK tyrosine phosphorylation. PDGF was shown to stimulate the tyrosine phosphorylation of p125FAK in porcine aortic endothelial (PAE) cells transfected with the wild type PDGF-beta receptors, but not in PAE cells transfected with PDGF-beta receptors in which the PI 3'-kinase binding sites (Tyr-740/751) were replaced by phenylalanine. PDGF-stimulated, PI 3'-kinase-dependent tyrosine phosphorylation of p125FAK was not inhibited by rapamycin, and thus it was dissociated from the activation of p70 S6 kinase, previously identified as a molecular downstream target of PI 3'-kinase. Thus, we have identified a PI 3'-kinase-dependent signal transduction pathway in the action of PDGF, which leads to the phosphorylation of p125FAK and paxillin.

3T3 Cells↗

PubMed1996

Induction of fibroblast growth factor receptor-1 mRNA and protein by platelet-derived growth factor BB.

Expression levels of growth factor receptors are subject to complex regulation, which is of consequence for their signaling capacity in physiological and pathological processes. We examined the regulation of expression levels of fibroblast growth factor receptor 1 (FGFR-1) in human fibroblasts treated with a panel of growth regulatory factors. Only platelet-derived growth factor BB (PDGF-BB) treatment had a significant effect and induced FGFR-1 mRNA levels fourfold, with a peak around 8 h of stimulation. The increase in mRNA levels was followed by an increased synthesis of FGFR-1 protein, which responded to basic FGF (bFGF) stimulation with induction of kinase activity and biological signaling. Thus, murine brain endothelial cells displayed an augmented induction of plasminogen activator activity in response to bFGF, following treatment with PDGF-BB. These data suggest that PDGF-BB could support FGFR-1-mediated biological responses in processes such as angiogenesis.

Animals↗

PubMed1996

Structural determinants in the platelet-derived growth factor alpha-receptor implicated in modulation of chemotaxis.

Activation of the platelet-derived growth factor (PDGF) beta-receptor leads to cell growth and chemotaxis. The PDGF alpha-receptor also mediates a mitogenic signal, but fails to induce cell migration in certain cell types. To examine this difference in signal transduction, a series of point-mutated PDGF alpha-receptors were analyzed. Porcine aortic endothelial cells expressing mutant PDGF alpha-receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type alpha-receptors and mutant alpha-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate. All mutant receptors were mitogenically active and their capacity to activate phosphatidylinositol 3'-kinase and phospholipase C-gamma was not different from that of the wild-type receptor. Tyr-768 was found to be phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was a considerable increase in phosphorylation of Ser-767. Tyr-993 was not phosphorylated, but mutation of this tyrosine residue to a phenylalanine residue resulted in increased efficiency of phosphorylation on Tyr-988. Tyr-1018 is known to be an autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind signal transduction molecules involved in negative modulation of the chemotactic signaling capacity, whereas phosphorylated Tyr-988 may mediate increased chemotaxis. Thus our data indicate that the PDGF alpha-receptor has an intrinsic ability to transduce a chemotactic signal, and that this signal is counteracted by overriding negative signals.

Amino Acid Sequence↗

PubMed1996

Mechanism of action of platelet-derived growth factor.

More than 20 years ago, platelet-derived growth factor (PDGF) was identified and later purified. Through recent years of intense research, a large body of information has been collected on how PDGF transduces its biological effects to responding cells. Two homologous receptors, the PDGF alpha- and beta-receptors, have been identified, which are receptor tyrosine kinases. Binding of PDGF leads to activation of the kinase and autophosphorylation. Particularly in the PDGF beta-receptor, a considerable number of autophosphorylation sites have been identified, which allow for physical interaction with signal transduction molecules. The signal transduction molecules are often enzymes, which undergo activity changes in conjunction with binding to the receptor. Other signal transduction molecules function as adaptors, which can couple to subunits equipped with catalytic activity. Through the activity changes of inherent or directly coupled catalytic activities, a signal is propagated, which ultimately results in a cellular response. PDGF is known to induce migration, proliferation and differentiation of different cells types. An array of signal transduction molecules has been shown to interact with the PDGF beta-receptor; several appear to contribute to the generation of the proliferative response, indicating the existence of parallel pathways for this response, which are utilized by many different growth factor receptors. Migration of cells towards PDGF appears to be more strictly dependent on activation of phosphatidylinositol 3' kinase. Interestingly, the PDGF alpha-receptor emits negative signals that inhibit simultaneous positive signals for migration induced by this receptor, or by other receptors, such as the PDGF beta-receptor. Virtually nothing is known about signal transduction initiated by PDGF, which generates differentiation responses. Since PDGF appears to play a role in different physiological and pathological processes, it is important to continue delineation of signal transduction pathways initiated through activation of the PDGF receptors.

Animals↗

PubMed1996

Expression of growth factors and growth factor receptors in capillary hemangioblastoma.

To elucidate the mechanisms underlying the regulation of growth and differentiation of capillary hemangioblastoma we studied the expression of selected growth factors and growth factor receptors by immunocytochemistry. As stromal cells of capillary hemangioblastoma express high levels of vascular endothelial growth factor (VEGF) and placental growth factor (P1GF) mRNA, we studied the distribution of the corresponding VEGF and P1GF proteins. We also studied the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptors (PDGFR) because their ligands have been reported to promote angiogenesis. The stromal cells expressed abundant EGFR and, in addition, some stromal cells expressed PDGFR-alpha but not PDGFR-beta. In contrast, the endothelial cells co-expressed PDGFR-alpha and PDGFR-beta. VEGF and P1GF were expressed by scattered stromal cells; however, more intense staining was observed in the endothelial cells of the intratumoral blood vessels, possibly indicating the secreted growth factors bound to their target receptors. We conclude that capillary hemangioblastomas express a variety of growth factor receptors and ligands, potentially involved in both autocrine and paracrine loops. The uniformly high EGFR expression is unique among brain tumors and may be associated with the typical morphology of capillary hemangioblastoma. The expression of highly angiogenic growth factors and their receptors may contribute to the rich vascularity of this enigmatic tumor.

Adult↗

PubMed1996

Fibroblast growth factor receptor 1-induced differentiation of endothelial cell line established from tsA58 large T transgenic mice.

Angiogenesis of capillary endothelial cells includes at least four sequential cellular responses: digestion of basement membrane, migration, proliferation, and differentiation. To study differentiation of endothelial cells, we established a brain capillary endothelial cell line from H-2Kb-tsA58 transgenic mice. These cells are stable at 33 degrees C and display endothelial cell-specific characters, such as expression of von Willebrand factor and binding sites for the lectin Bandeiraea simplifolia, and uptake of acetylated-low density lipoprotein. We measured the effects of a panel of growth factors on cellular responses. A number of factors, such as hepatocyte growth factor, vascular endothelial growth factor, and platelet-derived growth factor (PDGF)-AA failed to induce biological responses. PDGF-BB, epidermal growth factor, and acidic and basic fibroblast growth factor (FGF) induced proliferation of the cells. Of all the factors tested, only acidic FGF and basic FGF induced differentiation of the cells, visualized as the formation of tube-like structures of cells grown in three-dimensional collagen gels. All factors were also analyzed for their effects on plasminogen activator (PA)-induction and migration of the cells. Transfected cells, expressing a chimeric receptor, composed of the extracellular part from the PDGF alpha-receptor and the intracellular part from FGF receptor-1, responded to PDGF-AA treatment with plasminogen activator induction, migration, proliferation, and tube formation in collagen. These results indicate that FGF receptor-1 coupled to signal transduction pathways, leading to differentiation. This novel cell model offers the potential of detailed dissection of signal transduction pathways involved in the differentiation of endothelial cells.

Animals↗

PubMed1995

Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells.

A major pathway for mitogenicity is gated via the small GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras through the Src homology 2 (SH2) domain protein Grb2. The activated fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts did not bind Grb2 directly, but indirectly, through the small adaptor protein Shc, which was tyrosine-phosphorylated in response to fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in which Tyr766, a known autophosphorylation site, was changed to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2 stimulation of mutant FGFR-1-expressing cells still allowed formation of complexes containing Shc, Grb2, and the nucleotide exchange factor Sos and mediation of a mitogenic signal. Another pool of Grb2 was found in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2 stimulation. Stimulation with other growth factors did not lead to tyrosine phosphorylation of p89. As shown by "far-Western" analysis, p89 bound directly to the Grb2 SH2 domain, and this interaction was inhibited by a peptide containing the Y(P)-X-N motif. Tyrosine-phosphorylated p89 was found exclusively in the membrane fraction, indicating its role in bringing Grb2, as well as Sos, to the plasma membrane. These data support the concept of growth factor-specific coupling of Grb2 to the Ras pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Ligand-induced polyubiquitination of receptor tyrosine kinases.

The platelet-derived growth factor beta-receptor undergoes polyubiquitination as a consequence of ligand binding. In the present study, we have examined the ligand-induced receptor ubiquitination also in the other receptor tyrosine kinase (structurally different) subfamilies by immunoblotting with anti-ubiquitin antiserum. In addition to the platelet-derived growth factor alpha- and beta-receptors, all the monomeric receptor tyrosine kinases examined, such as the receptors for epidermal growth factor (subfamily I), colony stimulating factor-1 (subfamily III), and fibroblast growth factor (subfamily IV), were found to be ubiquitinated after ligand stimulation. However, the insulin receptor (subfamily II), which is a tetrameric molecule, was not. These data suggest that the ligand-induced polyubiquitination of the receptor is a general phenomenon observed in most of the monomeric receptor tyrosine kinases.

3T3 Cells↗