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PubMed2001

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

BACKGROUND: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. RESULTS: The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. CONCLUSIONS: We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

3T3 Cells↗
PubMedDate not supplied

Hyperglycemia-induced alteration of vascular smooth muscle phenotype.

We have previously reported that high glucose stimulates osteopontin (OPN) expression through protein kinase C-dependent pathway, as well as the hexosamine pathway, in cultured rat aortic smooth muscle cells (SMC). The finding prompted us to study in vivo expression of OPN in diabetes mellitus. In the present study, we found by immunohistochemistry that medial layers of the carotid arteries of streptozotocin (STZ)-induced diabetic rats, as well as the forearm arteries of diabetic patients, stained positive with OPN antibodies, whereas the staining of control rats, as well as nondiabetic patients, was negative. We also found that OPN stimulated migration and enhanced platelet-derived growth factor (PDGF)-mediated DNA synthesis of cultured rat aortic SMC. OPN and PDGF synergistically activated focal adhesion kinase (FAK), as well as extracellular signal-regulated kinase (ERK), which seems to be a reason for OPN-induced enhancement of PDGF-mediated DNA synthesis. Taken together, our present results raise a possibility that OPN plays a role in the development of diabetic vascular complications.

Animals↗
PubMed1996

The degree of porosity influences the release of growth factors by healing polytetrafluoroethylene (PTFE) grafts.

OBJECTIVES: The aim of this study was to determine the influence of the degree of porosity on the release of growth factors (PDGF AA, PDGF BB, bFGF) by healing PTFE grafts. DESIGN AND SETTING: Laboratory animal study. MATERIALS: 1 cm long segments of non-reinforced PTFE grafts (30 microns fibril length, 2 mm internal diameter, 0.39 mm thick) were placed as an abdominal aortic interposition in Lewis rats. Fifteen grafts served as control (Group A; porous grafts); in eight rats (Group B; non porous grafts) the PTFE graft was completely wrapped by a non-porous plastic envelope. Animals were killed 4 weeks after surgery. OUTCOME MEASURES: The release of PDGF AA, PDGF BB and bFGF was assessed by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: The release of PDGF AA, PDGF BB and bFGF was statistically higher in porous grafts. The only histological difference between the two groups was that porous PTFE grafts were invaded by many tufts of capillaries from the surrounding tissue, whereas this phenomenon was absent in non porous PTFE grafts. CONCLUSIONS: The degree of porosity influences the release of growth factors by healing PTFE grafts. This fact may have implication in the endothelisation of PTFE grafts and in myointimal hyperplasia formation as well.

Animals↗
PubMed1997

Monocyte chemoattractant protein-1 induces monocyte recruitment that is associated with an increase in numbers of osteoblasts.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family of cytokines. The principal function of MCP-1 is thought to be the stimulation of monocyte recruitment. Monocyte products are potential regulators of bone cell activity. Growth factors produced by monocytes may stimulate bone formation, while cytokines such as IL-1 and IL-6 can induce bone resorption. To determine whether MCP-1 enhances recruitment of monocytes during bone healing, studies were carried out in which MCP-1 was applied to osseous sites in vivo. Changes in monocyte number were determined by immunohistochemistry using the antibody ED-1 specific for peripheral monocytic cells. The effect of MCP-1 on osteoblast number was determined by counting the number of alkaline phosphatase positive cells in close proximity to bone. For comparison, osteoblast number was also determined following stimulation with platelet-derived growth factor (PDGF)-BB plus IGF-1 in vivo. Results indicate that MCP-1 stimulated a large increase in monocyte recruitment compared to vehicle alone. An increase in monocytes induced by MCP-1 was associated with an increase in the number of osteoblasts lining the bone surface, although not to the same magnitude as a positive control, PDGF-BB, and IGF-1. These results indicate that MCP-1 induces the recruitment of monocytes to bone and suggest that the recruitment is associated with an increase in osteoblast number. This is likely to occur via indirect mechanisms, because MCP-1 did not directly enhance DNA synthesis in osteoblastic cells in vitro. Thus, activated mononuclear phagocytes may play an important role in osseous wound healing by stimulating proliferation of osteoblastic cells, presumably through the elaboration of growth factors.

Alkaline Phosphatase↗
PubMed2004

Signaling aptamer/protein binding by a molecular light switch complex.

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [Ru(phen)2(dppz)]2+. The method takes advantage of the sensitive luminescence signal change of [Ru(phen)2(dppz)]2+ intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [Ru(phen)2(dppz)]2+/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and alpha-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.

Aptamers, Nucleotide↗
PubMed2006

Signaling aptamers created using fluorescent nucleotide analogues.

A new approach to creating fluorescent signaling aptamers using fluorescent nucleotide analogues is presented. The fluorescence quantum yield of nucleotide analogues such as 2-aminopurine strongly depends on base stacking interactions when incorporated into double or single stranded DNA. This property is used to generate a binding-specific fluorescence signal. Aptamers for human alpha-thrombin, immunoglobulin E, and platelet-derived growth factor B were modified with fluorescent nucleotide analogues in positions that undergo conformational changes. The resulting signaling aptamers show a specific, binding-induced increase in the fluorescence signal of up to 30-fold. Conformation-changing positions in these aptamers were identified by screening a set of modified aptamer sequences that each included a fluorescent nucleotide analogue at a different position. The positions for these modifications were estimated by modeling the aptamer secondary structure. It is likely that this approach to producing fluorescent signaling aptamers is of general use for protein-binding aptamers because of their "induced fit" binding mechanism.

Aptamers, Nucleotide↗
PubMed1993

Disulfide bonds in recombinant human platelet-derived growth factor BB dimer: characterization of intermolecular and intramolecular disulfide linkages.

Interchain cystines of PDGF-BB dimer were characterized by Edman reaction and by SDS-PAGE analysis on the protein which was chemically cleaved at Trp-40. It was found that Cys-43 has a key role in dimer formation, asymmetrically cross-linked to a cysteine residue of another identical subunit. The remaining cystines participate in the intramolecular disulfide linkages. Pepsin digestion of PDGF-BB dimer generated several small peptides and one ubiquitous Cys-containing peptide. Sequence analyses of several Cys-containing peptides indicated the existence of three intramolecular disulfide linkages including Cys-16--Cys-60, Cys-49--Cys-97, and Cys-53--Cys-99. Two interchain disulfide bonds of Cys-43--Cys-52 between two subunits were deduced from the partial reduction and alkylation of PDGF-BB. This study provides chemically determined disulfide linkages of PDGF-BB.

Acetylation↗
PubMed2005

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain.

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.

Amides↗
PubMed1999

Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor.

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.

3T3 Cells↗
PubMed2002

Potent and selective inhibitors of platelet-derived growth factor receptor phosphorylation. 1. Synthesis, structure-activity relationship, and biological effects of a new class of quinazoline derivatives.

A new series of 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives were found to show potent and selective inhibition of platelet-dervied growth factor (PDGF) receptor phosphorylation. In this exploration of the structure-activity relationships (SARs) of the prototype inhibitor KN1022, the 4-nitrophenylurea moiety was probed. We found that 4-substitution on the phenyl ring was optimal and the introduction of more than two substituents on the phenyl ring decreased activities. Bulky substituents on the phenyl ring enhanced activities. Thiourea analogues were also prepared, and the SARs were found to be slightly different from those of the urea derivatives. Through this research, we obtained some potent KN1022 derivatives such as 4-(4-methylphenoxy)phenyl (36, IC(50) 0.02 micromol/L), 4-tert-butylphenyl (16, IC(50) 0.03 micromol/L), and 4-phenoxyphenyl (21, IC(50) 0.08 micromol/L) analogues, which had almost a 10-fold increase in activity against KN1022. These potent compounds retained their high selectivity against the PDGF receptor family similar to KN1022. We also observed that these compounds could inhibit the PDGF-BB-induced proliferation of porcine vascular smooth muscle cells without cell toxicity almost at the same IC(50) values observed for PDGF receptor phosphorylation. To evaluate the biological effects in vivo, we selected some analogues on the basis of the measurement of the plasma drug concentration after oral administration to rats. Oral administration of the 4-chlorophenyl (6), 4-bromophenyl (9), or 4-isopropoxyphenyl (20) analogue to Sprague-Dawley rats (30 mg/kg, twice daily) resulted in significant inhibition (24-38%) of neointima formation in the carotid artery of the balloon catheter deendothelialized vessel in the rats. Therefore, 4-[4-(N-substituted carbamoyl)-1-piperazinyl]-6,7-dimethoxyquinazoline derivatives, which are potent inhibitors of PDGFR phosphorylation, may be expected to represent a new therapeutic approach for the treatment of various aspects of atherosclerosis and other cellular proliferative disorders.

Administration, Oral↗
PubMed2005

(6,7-Dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamines: platelet-derived growth factor receptor tyrosine kinase inhibitors with broad antiproliferative activity against tumor cells.

A series of (6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamines has been optimized to preserve both potent kinase inhibition activity against the angiogenesis target, the receptor tyrosine kinase of Platelet-Derived Growth Factor-BB (PDGF-BB), and to improve the broad tumor cell antiproliferative activity of these compounds. This series culminates in the discovery of 17 (JNJ-10198409), a compound with anti-PDGFR-beta kinase activity (IC(50)=0.0042 microM) and potent antiproliferative activity in six of eight human tumor cell lines (IC(50) < 0.033 microM).

Antineoplastic Agents↗
PubMed1998

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: attenuation of MAP kinase activation by cAMP, parathyroid hormone and forskolin.

The mitogen-activated protein (MAP) kinases (p44mapk and p42mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44mapk/ERK1 and p42mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay using myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.

Animals↗
PubMed1998

The mitogen-activated protein kinase pathway contributes to vanadate toxicity in vascular smooth muscle cells.

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC50: 30 microM). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.

Animals↗
PubMed2001

Association of ErbB2 Ser1113 phosphorylation with epidermal growth factor receptor co-expression and poor prognosis in human breast cancer.

The carboxyterminal domain of the epidermal growth factor receptor (EGFR)--a putative binding site for the ubiquitin ligase Cbl--is the site of serine phosphorylation events which are essential for ligand-dependent EGFR desensitization and degradation. Using a monoclonal antibody (aPS1113) which selectively recognizes the homologous phosphorylated domain in the ErbB2 oncoprotein, we show here that wild-type ErbB2 becomes Ser1113-phosphorylated following treatment of 3T3 cells with growth factors or tyrosine phosphatase inhibitors. In EGFR-overexpressing A431 cells, ligand-inducible aPS1113 immunoreactivity declines more rapidly than other detectable phosphorylation events and is followed by EGFR downregulation. Analysis of 65 ErbB2-expressing primary breast cancers reveals a highly significant relationship between Ser1113 phosphorylation and EGFR overexpression (p < 0.0001) as well as an association with poor prognosis (p = 0.005). We submit that ErbB2 Ser1113 phosphorylation status represents a novel and informative biomarker of cancer cell biology and tumor behavior.

Animals↗
PubMed2001

Arsenite inhibits Ras-dependent activation of ERK but activates ERK in the presence of oncogenic Ras in baboon vascular smooth muscle cells.

Exposure to arsenical compounds enhances the risk of atherosclerosis. The reason is unknown but it might be because an effect of arsenite (As3+) on plaque smooth muscle cells (SMCs) activation of extracellular signal-regulated kinase (ERK), a crucial mediator of SMC function. We found that arsenite inhibits the activation of ERK by platelet-derived growth factor-BB (PDGF-BB). This inhibitory effect depends on the time of arsenite exposure, is reversible, and is attenuated by preincubation of SMCs with the antioxidant N-acetyl-cysteine. These observations are consistent with the assumption that oxidative stress is involved. The blockade of ERK by arsenite may be mediated by an inhibition of Ras as arsenite prevents GTP-loading of Ras in response to PDGF-BB. Moreover, the Ras blockade by arsenite is not specific for PDGF-BB because it was also observed following stimulation of SMCs with EGF. To address the role of Ras, we expressed constitutively active, GTP-bound Ha-Ras (V12Ras). Unexpectedly, in V12Ras expressing-SMCs, arsenite stimulates ERK, but still decreases ERK activity in the presence of PDGF-BB. Our data suggest that arsenite inhibits the Ras/ERK pathway in SMCs, and that arsenite may activate ERK in Ras-transformed cells by mechanisms different from those employed by growth factors.

Animals↗
PubMed2001

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1998

Keratinocyte growth factor decreases pulmonary edema, transforming growth factor-beta and platelet-derived growth factor-BB expression, and alveolar type II cell loss in bleomycin-induced lung injury.

Keratinocyte growth factor (KGF), a potent growth factor for type II pneumocytes and Clara cells, has been shown to prevent the end-stage pulmonary fibrosis and mortality in a rat model of bleomycin-induced lung injury. In this study, protective effects of KGF were explored during the earlier course of bleomycin-induced lung injury by studying protein exudation in alveolar edema fluids, pulmonary expression of transforming growth factor-beta (TGF beta) and platelet-derived growth factor-BB (PDGF-BB), and changes in type II pneumocytes and Clara cells after i.t. (intratracheal) bleomycin injection following KGF- or saline-pretreatment in rats. Total protein in bronchoalveolar lavage (BAL) fluids after bleomycin injury from KGF-pretreated rats was significantly lower than the levels in saline-pretreated rats. TGF beta protein in BAL fluids which peaked at day 3 after i.t. bleomycin in saline-pretreated lungs was not significantly increased at any time points in KGF-pretreated rats. PDGF-BB protein in whole lung tissues of KGF-pretreated rats also remained near normal throughout the course after i.t. bleomycin, in contrast to the significant increase in saline-pretreated rats. Numbers of type II pneumocytes and Clara cells in KGF-pretreated lungs after a high dose of bleomycin were close to the normal in intact lungs. At the same dose of bleomycin injury, type II pneumocytes in saline-pretreated lungs were markedly decreased, while the number of Clara cells in these rats was relatively preserved as the pre-injury level. In conclusion, KGF prevents bleomycin-induced end-stage pulmonary injury and mortality probably at least partly by decreasing protein-rich pulmonary edema, protein expression of fibrogenic cytokines TGF beta and PDGF-BB, and type II cell loss during the course of lung injury.

Animals↗