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PubMed2001

Polymeric system for dual growth factor delivery.

The development of tissues and organs is typically driven by the action of a number of growth factors. However, efforts to regenerate tissues (e.g., bone, blood vessels) typically rely on the delivery of single factors, and this may partially explain the limited clinical utility of many current approaches. One constraint on delivering appropriate combinations of factors is a lack of delivery vehicles that allow for a localized and controlled delivery of more than a single factor. We report a new polymeric system that allows for the tissue-specific delivery of two or more growth factors, with controlled dose and rate of delivery. The utility of this system was investigated in the context of therapeutic angiogenesis. We now demonstrate that dual delivery of vascular endothelial growth factor (VEGF)-165 and platelet-derived growth factor (PDGF)-BB, each with distinct kinetics, from a single, structural polymer scaffold results in the rapid formation of a mature vascular network. This is the first report of a vehicle capable of delivery of multiple angiogenic factors with distinct kinetics, and these results clearly indicate the importance of multiple growth factor action in tissue regeneration and engineering.

Actins↗
PubMed2003

Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2.

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.

Animals↗
PubMed1999

Stimulation of tumour growth by wound-derived growth factors.

The goal of this work was to determine the molecular basis for the induction of tumour vascularization and progression by injury. Magnetic resonance imaging (MRI) studies demonstrated that administration of wound fluid derived from cutaneous injuries in pigs reduced the lag for vascularization and initiation of growth of C6 glioma spheroids, implanted in nude mice, and accelerated tumour doubling time. The former effect can be attributed to the angiogenic capacity of wound fluid as detected in vivo by MRI, and in vitro in promoting endothelial cell proliferation. The latter effect, namely the induced rate of tumour growth, is consistent with the angiogenic activity of wound fluid as well as with the finding that wound fluid was directly mitogenic to the tumour cells, and accelerated growth of C6 glioma in spheroid culture. Of the multiple growth factors present in wound fluid, two key factors, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and platelet-derived growth factor (PDGF), were identified as the dominant mitogens for C6 glioma, and inhibition of their activity using specific neutralizing antibodies suppressed the mitogenic effect of wound fluid on DNA synthesis in C6 glioma. This study suggests that the stimulatory effect of injury on tumour progression can possibly be attenuated by therapeutic targeting directed against a limited number of specific growth factors.

Angiogenesis Inducing Agents↗
PubMed1997

Homocysteine as a modulator of platelet-derived growth factor action in vascular smooth muscle cells: a possible role for hydrogen peroxide.

1. Homocysteine is an independent risk factor for cardiovascular disease. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. Therefore, we examined the effect of homocysteine on cell replication of rat cultured vascular smooth muscle cells (VSMCs) at concentrations similar to those observed in clinical studies. 2. The incorporation of [3H]-thymidine was used as a marker of mitosis. Homocysteine (250-500 microM) was a weak mitogen as compared to platelet-derived growth factor-BB (PDGF-BB, 1 nM) and serum (10%), but it potentiated the mitogenic effect of PDGF-BB four fold at 500 microM. This enhancement of mitogenesis was blunted by the addition of the scavenging enzyme catalase or the antioxidant N-acetyl-L-cysteine. 3. Furthermore, stimulation of VSMC with homocysteine (25-500 microM) decreased the glutathione peroxidase activity of the cells to 50% of control at 500 microM. Inversely, homocysteine enhanced the superoxide dismutase (SOD) activity to 137% of control at 500 microM, but it had no effect on the catalase activity. 4. Homocysteine decreased the activity of bovine purified liver cytosolic glutathione peroxidase in a time- and dose-dependent manner. The maximum decrease was 50%. 5. In summary, homocysteine has a weak mitogenic effect on VSMC, but it dramatically enhances the mitogenic response of PDGF-BB, presumably by disturbing the activity of antioxidant enzymes.

Animals↗
PubMed1998

Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors.

1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.

Animals↗
PubMed2000

Inhibitory mechanism of tranilast in human coronary artery smooth muscle cells proliferation, due to blockade of PDGF-BB-receptors.

We have previously reported that tranilast, an anti-allergic drug, prevented the experimental intimal thickening in the rat and mouse femoral arteries and its effect may be exerted through the inhibition of vascular smooth muscle cell proliferation. However, its inhibitory mechanism has yet to be understood. In this study, we investigated the inhibitory effect of tranilast on platelet-derived growth factor BB-homodimer (PDGF-BB) mediated signal transduction pathways in cultured human coronary artery smooth muscle cells (CASMCs). Growth responses to PDGF-BB were measured by [(3)H]-thymidine incorporation or cell counting. Activation of DNA synthesis and augmentation of cell proliferation stimulated by PDGF-BB in quiescent cultures of CASMCs were inhibited by tranilast in a concentration-dependent manner. Western blot analysis of lysates from CASMCs with an anti-activated mitogen-activated protein (MAP) kinase antibody revealed that tranilast (10 - 300 microM) inhibited MAP kinase activation by PDGF-BB in a concentration-dependent manner. Tranilast also reduced PDGF-BB-stimulated tyrosine phosphorylation of a 180 kDa band, corresponding in mass to the PDGF beta-receptor, as shown by immunoblots using an anti-phosphotyrosine antibody. Receptor-binding study with [(125)I]-PDGF-BB on CASMCs showed that tranilast (10 - 1000 microM) inhibited the specific binding of PDGF-BB to cell surface receptors in a concentration-dependent manner. Scatchard analysis revealed that pretreatment with 300 microM tranilast decreased the maximum binding capacity (B(max)) from 27.6 to 18.0 fmol 10(6) cells(-1) without affecting binding affinity (K(d) approximately 0.15 nM), indicating a non-competitive inhibition of the receptor binding. These results suggest that the suppression of human CASMC growth by tranilast might be at least partly due to blockade of PDGF-BB-receptor binding.

Animals↗
PubMed2000

Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis.

The second messenger cyclic AMP regulates diverse biological processes such as cell morphology and cell growth. We examined the role of the second messenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morphology and the intracellular transduction pathway mediated by platelet-derived growth factor beta-receptor (PDGF-Rbeta). The effect of PDGF-BB on VSMCs growth was assessed by [(3)H]-thymidine incorporation. Tyrosine phosphorylation of PDGF-Rbeta, PLC-gamma1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm alpha-actin filaments, paxillin and PDGF-Rbeta was performed by immunfluorescence microscopy. Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increase in the cyclic AMP concentration after 2 h which correlated with change of VSMC morphology including complete disruption of the Sm alpha-actin filament array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein. Maximal decrease of 70% was observed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRNA level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesis although PDGF-Rbeta level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine phosphorylation of the PDGF-Rbeta, PI 3'-K, PLC-gamma1 and ERK1/2 indicating an action of cyclic AMP on PDGF-beta receptor. We conclude that although cyclic AMP attenuates the PDGF-Rbeta mediated intracellular transduction pathway, an intact actin filament may be required for the PDGF-BB-induced DNA synthesis in VSMCs.

Actins↗
PubMed2001

Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase.

1. Arginine-specific ADP-ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP-ribose from NAD(+) to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline-inducible expression of human ART. 2. From a panel of growth factors, platelet-derived growth factor-BB (PDGF-BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF-AA was not a substrate. Under conditions of maximum labelling 5 mol ADP-ribose was incorporated per mol of PDGF-BB. 3. Purified (ADP-ribosyl)-PDGF-BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF-BB. 4. PDGF-dependent [(3)H-methyl]-thymidine incorporation was measured in the ART1-transfected fibroblast cell line at physiological concentrations of PDGF-BB, and without addition of extracellular NAD(+). Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF-BB, but not to PDGF-AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). 5. In conclusion, we propose that PDGF-BB-dependent signalling may be regulated by post-translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1-transfected fibroblasts.

Animals↗
PubMed2005

Effect of serum withdrawal on the contribution of L-type calcium channels (CaV1.2) to intracellular Ca2+ responses and chemotaxis in cultured human vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L-type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle alpha-actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin-coated plates was also quantified. The number and affinity of dihydropyridine-binding sites was assessed using [5-methyl-3H]PN 200-110 binding. In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet-derived growth factor-BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF-induced migration. In serum-deprived cells, smooth muscle alpha-actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF-induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF-induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum-deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF-induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum-deprived hVSMC. Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine-binding sites (B(max)) and a decrease in affinity (K(D)). Serum deprivation of hVSMC results in increased expression of smooth muscle alpha-actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

ATP and adenosine prevent via different pathways the activation of caspases in apoptotic AKR-2B fibroblasts.

Confluent AKR-2B fibroblasts rapidly disintegrate after serum deprivation.27 ATP or adenosine added immediately after serum removal afforded substantial protection against cell death even for a long period of 24 h. ED50 values were 14 and 110 microM for ATP and adenosine, respectively. In the presence of 5 microg/ml cycloheximide the protective effect of both substances was suppressed, indicating that protein synthesis is required. The protective effect of ATP was highly specific since among numerous tested derivatives only ATP-[gamma-S] exhibited a substantial protective effect. The ability of ATP and adenosine to modulate cell division was analyzed. Both substances did not exhibit any mitogenic effect. Adenosine completely blocked PDGF-BB induced cell division, whereas ATP had no effect. Unlike adenosine, ATP strongly stimulated Ca2+-release from intracellular stores. On the other hand, adenosine stimulated an increase in the intracellular concentration of cAMP from 0.4 - 1.5 microM, whereas ATP decreased the content below 0.1 microM. ATP stimulated the phosphorylation of MAP-kinase, RSK and p70S6-kinase; adenosine was inactive. After complexation of [Ca2+]i the protective effect of ATP was greatly lost while adenosine was still active. Surprisingly neither ATP nor adenosine caused an activation of PKC-isoforms. After incubation with pertussis toxin, the protection by ATP was reduced indicating an involvement of Gi-proteins in the signal transduction induced by ATP. Our results indicate that ATP as well as adenosine are potent inhibitors of cell death caused by serum deprivation and that this protective effect apparently occurs via distinct pathways. However, both pathways must converge at the point of caspase activation, since the stimulation of DEVDase- and VEIDase-activities, respectively, are suppressed by either ATP or adenosine.

Adenosine↗
PubMed2006

PKCdelta clustering at the leading edge and mediating growth factor-enhanced, but not ecm-initiated, dermal fibroblast migration.

We have previously shown that the immobilized extracellular matrices (ECMs) initiate cell migration and soluble growth factors (GFs) further enhance ECM-initiated cell migration. GFs alone cannot initiate cell migration. To further investigate the specificity of the two signaling mechanisms, we focused on the protein kinase C (PKC) family genes in primary human dermal fibroblasts (DFs). We here show that platelet-derived growth factor-BB (PDGF-BB) strongly stimulates membrane translocation and leading edge clustering of protein kinase Cdelta (PKCdelta). In contrast, attachment to collagen matrix alone does not cause the translocation. Although the kinase function of PKCdelta is dispensable for initial membrane translocation, it is critical for its sustained presence at the cells's leading edge. Blockade of endogenous PKCdelta signaling with dominant-negative kinase-defective PKC (PKCdelta-KD) or PKCdelta-small interfering RNA (siRNA) completely inhibited PDGF-BB-stimulated DF migration. In contrast, neither PKCdelta-KD nor PKCdelta-siRNA affected collagen-induced initiation of DF migration. Overexpression of a constitutively activated PKCdelta (PKCdelta-R144/145A) partially mimics the effect of PDGF-BB. However, PKCdelta-KD, PKCdelta-siRNA, or PKCdelta-R144/145A does not affect PDGF-BB-stimulated activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase1/2, or c-Jun N-terminal kinase. Instead, inhibition of PKCdelta blocks PDGF-BB-stimulated activation of signal transducer and activator of transcription 3 (Stat3). This study unveiled the specificity of PKCdelta in the control of DF migration.

Becaplermin↗
PubMed2006

Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.

Angiogenesis Inducing Agents↗
PubMed2002

Bis(1H-2-indolyl)-1-methanones as inhibitors of the hematopoietic tyrosine kinase Flt3.

Aberrant expression and activating mutations of the class III receptor tyrosine kinase Flt3 (Flk-2, STK-1) have been linked to poor prognosis in acute myeloid leukemia (AML). Inhibitors of Flt3 tyrosine kinase activity are, therefore, of interest as potential therapeutic compounds. We previously described bis(1H-2-indolyl)-1-methanones as a novel class of selective inhibitors for platelet-derived growth factor receptors (PDGFR). Several bis(1H-2-indolyl)-1-methanone derivatives, represented by the compounds D-64406 and D-65476, are also potent inhibitors of Flt3. They inhibit proliferation of TEL-Flt3-transfected BA/F3 cells with IC(50) values of 0.2-0.3 microM in the absence of IL-3 but >10 microM in the presence of IL-3. Ligand-stimulated autophosphorylation of Flt3 in EOL-1 cells and corresponding downstream activation of Akt/PKB are effectively inhibited by bis(1H-2-indolyl)-1-methanones whereas autophosphorylation of c-Kit/SCF receptor or c-Fms/CSF-1 receptor is less sensitive or insensitive, respectively. Flt3 kinase purified by different methods is potently inhibited in vitro, demonstrating a direct mechanism of inhibition. 32D cells, expressing a constitutively active Flt3 variant with internal tandem duplication are greatly sensitized to radiation-induced apoptosis in the presence of D-64406 or D-65476 in the absence but not in the presence of IL-3. Thus, bis(1H-2-indolyl)-1-methanones are potential candidates for the treatment of Flt3-driven leukemias.

Animals↗
PubMed1997

Cells transformed by ODC, c-Ha-ras and v-src exhibit MAP kinase/Erk-independent constitutive phosphorylation of Sos, Raf and c-Jun activation domain, and reduced PDGF receptor expression.

While it is known that the constitutive activity of a variety of signal transduction molecules leads to cell transformation, a key unresolved question is whether these wirings converge to a common intermediate(s) that dictates transformation. In this study, we investigated whether NIH3T3 and Rat-1 cells transformed by human ornithine decarboxylase (ODC), c-Ha-rasVal12 and temperature-sensitive v-src oncogene display common alteration(s) in the components that relay PDGF-mediated signals in normal fibroblasts. The ras- and ODC-transformed cells did not show constitutively elevated tyrosine phosphorylation of the phospholipase Cgamma-1 (PLCgamma-1), RasGTPase-activating protein (GAP), phosphotyrosine phosphatase Syp, Shc proteins, and phosphatidylinositol 3-kinase (PI3-K) or activation of the MAP kinase (Erk1 and Erk2), p70 S6 kinase or the Janus protein tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) protein-1 pathways. Instead, the Ras nucleotide exchange factor Sos-1 and Raf-1 kinase exhibited constitutive phosphorylations, as deduced from their electrophoretic mobility shifts in polyacrylamide gels. Hence a kinase distinct from Erk1 and Erk2, previously known to feedback phosphorylate Sos-1 and Raf-1, is responsible for the phosphorylation of these molecules in the transformants. We also demonstrate that the ras- and ODC-transformed cells exhibit loss of both the PDGF alpha- and beta-receptors, while the v-Src-transformants show a predominant reduction in the beta-receptors. Moreover, all the transformed cell lines were found to display a constitutive increase in phosphorylation of c-Jun on serines 63 and 73, which appears to be governed by an as yet unknown kinase.

3T3 Cells↗
PubMed1998

Identification of Tyr-762 in the platelet-derived growth factor alpha-receptor as the binding site for Crk proteins.

Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) alpha-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, affinity purification using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the purified proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF alpha-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was specifically inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF alpha-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type alpha-receptor. In contrast, the Y762F mutant PDGF alpha-receptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF alpha-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF alpha-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF beta-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the beta-receptor, which is localized at an analogous position to Tyr-762 in the alpha-receptor, binds RasGAP. RasGAP is not bound to the alpha-receptor. Thus, this region in the kinase inserts of the two receptors may be important for the divergency in signaling from the two PDGF receptors.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Fibroblast growth factor receptor-1 mediates chemotaxis independently of direct SH2-domain protein binding.

Endothelial cells expressing fibroblast growth factor receptor-1 (FGFR-1) migrate and proliferate in response to treatment with FGF. We analysed ligand-induced migration and proliferation of porcine aortic endothelial cells expressing wild-type FGFR-1, point-mutated Y766F FGFR-1, unable to activate phospholipase C-gamma1 (PLC-gamma1), or carboxyl-terminally truncated FGFR-1, lacking either 48 (from amino acid 774 in the FGFR-1 sequence) or 63 (from amino acid 759) amino acid residues of the C-terminal tail. The truncated CT63 FGFR-1 mutant failed to mediate chemotaxis, but was in response to ligand stimulation capable of mediating proliferation of the cells, stimulation of MAP kinase activity and tyrosine phosphorylation of FRS2, an FGFR-1 specific signaling molecule. The defect in migration-capacity of CT63 was not due to loss of Y766, and thereby PLC-gamma1 activation, since cells expressing the mutant Y766F FGFR-1 migrated as efficiently as the wild-type receptor cells. Induction of phospholipase A2 (PLA2) activity by the activated FGFR-1 was dependent on the presence of Y766, and was therefore also not critical for the chemotactic response. Although the FGFR-1 only very inefficiently mediates activation of phosphatidylinositol 3' kinase (PI 3-kinase), the PI 3-kinase inhibitor wortmannin suppressed wild-type FGFR-1 mediated migration. We conclude that the signal transduction pathway for FGFR-1 mediated migration is independent of phosphotyrosine residues in the receptor and requires activation of a wortmannin-sensitive enzyme.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Downregulation of platelet-derived growth factor receptor-beta in Shp-2 mutant fibroblast cell lines.

The SH2-containing tyrosine phosphatase Shp-2 appears to function downstream of a variety of growth factor receptors and might play a positive role in cell proliferation. Here we report that expression of the beta subunit of platelet-derived growth factor receptor (PDGFR-beta) was specifically downregulated in mutant fibroblasts lacking a functional Shp-2, while the levels of PDGFR-alpha EGFR and IGFIR were not changed. PDGF-stimulated DNA synthesis and extracellular signal regulated kinase (Erk) activation was severely suppressed in mutant cells. RasGAP, that responds to activation of PDGFR-beta but not PDGFR-alpha, was not phosphorylated on tyrosine in mutant cells upon PDGF-treatment. Northern blot analysis failed to detect PDGFR-beta mRNA in mutant cells. The transcription initiation from the PDGFR-beta gene promoter was not significantly changed, but the half-life of its mRNA was shortened in Shp-2 mutant cells. These observations indicate that Shp-2 not only participates in transmission of signals from growth factor receptors but also plays a specific role in the control of the PDGFR-beta expression. We propose that this is an important mechanism for the positive control of cell proliferation by Shp-2.

Animals↗