Search PubMed⌕ Search

Biomedical subjects

I Zachary

Publications and source records attributed to I Zachary.

At least 37 records · Page 2Linked to original sources
PubMed1997

VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries.

Thickening of the arterial intima and smooth muscle cell (SMC) proliferation remain major problems after vascular surgery and other types of vascular manipulations. We studied the effect of endothelial cell (EC)-specific vascular endothelial growth factor (VEGF) gene transfer on the thickening of the intima using a silicone collar inserted around carotid arteries that acted both as the agent that caused intimal SMC growth and as a reservoir for the transfected gene. The model preserved EC integrity and permitted direct extravascular gene transfer without any intravascular manipulation. Compared to beta-galactosidase (lacZ)-transfected control arteries, plasmid/liposome-mediated VEGF gene transfer significantly reduced intimal thickening 1 week after the gene transfer. Administration to the experimental animals of the nitric oxide (NO) synthase inhibitor L-NAME abolished the difference in intimal thickening between VEGF and lacZ-transfected arteries. Furthermore, VEGF caused NO release from cultured human umbilical vein EC. It is concluded that extravascular VEGF gene transfer attenuates intimal growth and could be useful for the prevention of intimal thickening during vascular surgery. Our results further suggest that VEGF may reduce SMC proliferation via a mechanism that involves VEGF-induced NO production from the endothelium.

Animals↗
PubMed1997

Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells.

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of Mr 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and Mr 145,000, 120,000, 97,000, and 65,000-70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-gamma tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3'-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)), which was both rapid and concentration-dependent. VEGF produced similar effects on p125(FAK) in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125(FAK). Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125(FAK) tyrosine phosphorylation in HUVECs. The effect of VEGF on p125(FAK) tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125(FAK) tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125(FAK)/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125(FAK), paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125(FAK) and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.

Cell Adhesion Molecules↗
PubMed1997

Prolactin stimulates the JAK2 and focal adhesion kinase pathways in human breast carcinoma T47-D cells.

Treatment of T47-D human breast carcinoma cells with recombinant prolactin (rhPRL) induced a concentration- and time-dependent increase in the phosphotyrosine content of JAK2. rhPRL also stimulated JAK2 tyrosine phosphorylation more weakly in three other breast carcinoma lines, MCF-7, ZR-75-1 and MDA-MB-231. Furthermore it stimulated tyrosine phosphorylation of two isoforms of the transcriptional activator STAT5, STAT5a and STAT5b. Surprisingly, rhPRL treatment of T47-D cells also stimulated tyrosine phosphorylation of focal adhesion kinase (FAK), as determined by immunoprecipitation with anti-phosphotyrosine antibody followed by immunoblotting with a specific FAK antibody. The effect of rhPRL was rapid and concentration-dependent, being maximal at 5 ng/ml. At rhPRL concentrations above 25 ng/ml, FAK tyrosine phosphorylation declined but remained above control levels at 100 ng/ml. rhPRL also stimulated paxillin tyrosine phosphorylation in T47-D cells with similar concentration- and time-dependence. In a second human breast carcinoma cell line, MCF-7, rhPRL produced very similar effects on FAK and paxillin tyrosine phosphorylation. These findings identify a new protein tyrosine kinase pathway in the action of the lactogenic hormone rhPRL and represent the first report that a hormone acting through a member of the haemopoietin receptor superfamily can regulate the FAK/paxillin pathway.

Breast Neoplasms↗
PubMed1997

Focal adhesion kinase.

Focal adhesion kinase (FAK) is a member of a growing family of non-receptor protein tyrosine kinases. Though originally identified as a putative substrate for the oncogenic tyrosine kinase pp60v-src, it is now well-established that FAK tyrosine phosphorylation is induced by adhesion of cell surface integrins to extracellular matrix and by a variety of other extracellular factors including the ligands for receptor tyrosine kinases and for seven transmembrane domain G-protein-coupled receptors. FAK can associate with multiple cellular components including other focal adhesion-associated proteins and signalling molecules. FAK is localized to focal adhesions and is centrally implicated in the regulation of cell motility and adhesion. Knocking out the FAK gene in mice prevents normal embryonic development and is associated with loss of mesenchymal cell motility. Recent findings further suggest novel tissue-specific functions for FAK, particularly in the brain. These findings implicate FAK in the aberrant migration of cells in a variety of diseases and suggest that FAK may be a novel target for therapeutic strategies.

Animals↗
PubMed1995

Differential effects of platelet-derived growth factor BB on p125 focal adhesion kinase and paxillin tyrosine phosphorylation and on cell migration in rabbit aortic vascular smooth muscle cells and Swiss 3T3 fibroblasts.

In rabbit aortic vascular smooth muscle cells (VSMC) platelet-derived growth factor BB (PDGF-BB) stimulated the tyrosine phosphorylation of phospholipase C-gamma, p120 GTPase-activating protein, and the p85 alpha subunit of phosphatidylinositol 3'-kinase only at high concentrations (5-25 ng/ml). In contrast, PDGF-BB induced a rapid and concentration-dependent increase in p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, which was half-maximal and maximum at 1 and 2.5 ng/ml, respectively. Saliently, stimulation of p125FAK tyrosine phosphorylation was sustained at up to 100 ng/ml PDGF-BB and for prolonged times of treatment. With similar concentration dependence, PDGF-BB stimulated the tyrosine phosphorylation of the 68-kDa focal adhesion-associated protein, paxillin. PDGF-BB also induced p125FAK and paxillin tyrosine phosphorylation in human aortic VSMC. PDGF-BB caused no detectable disruption of the actin cytoskeleton in VSMC. PDGF-BB stimulated rabbit VSMC migration with a very similar concentration dependence to that for p125FAK and paxillin tyrosine phosphorylation. PDGF-BB was equally effective in stimulating p125FAK and paxillin tyrosine phosphorylation under conditions similar to those used for cell migration. In Swiss 3T3 fibroblasts, PDGF-BB and -AA stimulated p125FAK tyrosine phosphorylation and cell migration only at low concentrations, and stimulation was abolished at 10-25 ng/ml. PDGF-AA failed to stimulate tyrosine phosphorylation, mitogenesis, and chemotaxis in rabbit VSMC, and immunoblot analysis showed that rabbit VSMC expressed PDGF beta-receptors but no alpha-receptors. These results implicate p125FAK in the chemotactic response to PDGF-BB and suggest that the ability of PDGF-BB to trigger the p125FAK pathway may be dependent both upon cell type and receptor isotype expression.

3T3 Cells↗
PubMed1995

Signalling mechanisms in the regulation of vascular cell migration.

The migration of arterial vascular smooth muscle cells (VSMC) is thought to play a central role in atherogenesis and restenosis. The migration of several other cell types, including monocytes, T-lymphocytes and endothelial cells is also involved in the development of the mature atherosclerotic lesion. Several defined growth factors, cytokines and extracellular matrix components which are released at the sites of lesions have been implicated in the regulation of migration of VSMC and other lesion-associated cells. Platelet-derived growth factor BB-homodimer of PDGF (PDGF-BB) is strongly implicated in neo-intima formation in vivo and is the most potent known chemoattractant for VSMC in vitro. Dynamic interactions between cell surface adhesive receptors (integrins) for ECM components, organisation of the actin cytoskeleton and the turnover of focal adhesions are all key processes in cell locomotion and migration. The signal transduction pathways which mediate the chemotactic effects of PDGF-BB and other migration factors on VSMC are unknown, but several classes of cellular components are implicated including components associated with focal adhesions, small GTP-binding proteins of the rho family, and certain substrates of the PDGF beta-receptor. Tyrosine phosphorylation of the novel focal adhesion-associated protein tyrosine kinase, p125 focal adhesion kinase (p125FAK), is regulated by integrins and by several factors which alter actin cytoskeletal organisation. Recent findings suggest that tyrosine phosphorylation of p125FAK and other focal adhesion-associated proteins may be implicated in the chemotactic response of VSMC to PDGF-BB. The migratory response to PDGF-BB may be dependent on both ligand isoform bio-availability and on receptor-isotype expression as well as on down-stream signalling events. Ultimately, cell migration in vivo will be determined by a complex array of diverse extracellular molecules organised in intercellular paracrine/autocrine networks as well as multiple interacting intracellular signal transduction pathways.

Arteriosclerosis↗
PubMed1993

Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with bombesin caused a striking increase (21-fold) in the tyrosine phosphorylation of the cytoskeleton-associated protein paxillin, as judged by anti-phosphotyrosine Western blots of anti-paxillin immunoprecipitates. Vasopressin and endothelin also stimulated paxillin tyrosine phosphorylation. Bombesin-stimulated tyrosine phosphorylation of paxillin was detectable within 1 min and was concentration-dependent (half-maximum effect at 0.09 nM). Bombesin stimulation of paxillin tyrosine phosphorylation could be dissociated from both protein kinase C (PKC) activation and the mobilization of Ca2+ from intracellular stores. Activation of PKC in quiescent Swiss 3T3 cells using the tumor promoter phorbol 12,13-dibutyrate (PDB) increased the tyrosine phosphorylation of paxillin in a time-dependent manner but was less effective than bombesin and stimulated detectable phosphorylation only within 5 min, considerably slower than bombesin-induced tyrosine phosphorylation of paxillin. Furthermore, the selective PKC inhibitor, GF109203X, or down-regulation of PKC using prolonged treatment with PDB markedly inhibited the stimulation of paxillin tyrosine phosphorylation by PDB but had little effect on the response to bombesin. In contrast, cytochalasin D, an agent that selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced paxillin tyrosine phosphorylation. This is the first report to identify paxillin as a substrate for neuropeptide-stimulated tyrosine phosphorylation.

3T3 Cells↗
PubMed1993

Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation. Role of protein kinase C, Ca2+ mobilization, and the actin cytoskeleton.

Activation of protein kinase C (PKC) in quiescent Swiss 3T3 cells using either the tumor promoter phorbol 12,13-dibutyrate (PDB) or diacylglycerols increased the tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) by 3.8-fold. PDB stimulation of p125FAK tyrosine phosphorylation was detected within 1 min and reached a maximum within 5 min, considerably slower than PDB stimulation of 80K/MARCKS phosphorylation which was maximal within 1 min. In sharp contrast, bombesin-induced tyrosine phosphorylation of p125FAK reached a maximum (8-fold stimulation) within 1 min after addition of the peptide and occurred with a half-maximal effect of 0.08 nM, 6-fold lower than the half-maximal effect of bombesin on 80K/MARCKS phosphorylation. Down-regulation of PKC by prolonged treatment with PDB blocked the effect of PDB on p125FAK tyrosine phosphorylation but had no effect on the response to bombesin. A selective inhibitor of PKC, GF 109203X, markedly inhibited the stimulation of p125FAK tyrosine phosphorylation by PDB but had little effect on the response to bombesin, vasopressin, and endothelin. Bombesin stimulation of tyrosine phosphorylation could also be dissociated from mobilization of Ca2+ from intracellular stores. Depletion of the intracellular Ca2+ pool by treatment with the tumor promoter thapsigargin completely blocked the ability of bombesin to transiently increase the cytosolic Ca2+ concentration but had no effect on bombesin stimulation of p125FAK tyrosine phosphorylation. In contrast, cytochalasin D, an agent which selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced p125FAK tyrosine phosphorylation. Within the same concentration range (0.3-2 microM), the drug had no effect on other early events stimulated by bombesin, including Ca2+ mobilization and activation of PKC. These findings demonstrate that neither the PKC nor Ca2+ pathways are responsible for the rapid stimulation of p125FAK tyrosine phosphorylation by neuropeptide growth factors. Furthermore, the integrity of the actin cytoskeleton is essential for the effects of both PDB and bombesin.

3T3 Cells↗
PubMed1992

Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate.

Neuropeptide-stimulated tyrosine phosphorylation of specific components in Swiss 3T3 cells was investigated using monoclonal antibodies directed against the src transformation-associated substrates p125 focal adhesion kinase (FAK), a novel type of cytosolic tyrosine kinase, and p130. Treatment of Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, and endothelin caused a striking increase in the tyrosine phosphorylation of p125FAK, as judged either by anti-phosphotyrosine (anti-Tyr(P)) Western blots of anti-p125FAK immunoprecipitates, or by anti-p125FAK immunoblots of anti-Tyr(P) immunoprecipitates. Bombesin-stimulated tyrosine phosphorylation of p125FAK was detectable within seconds and concentration-dependent (half-maximum effect of 0.3 nM). Neuropeptides also stimulated the tyrosine phosphorylation of a second component of M(r) 130,000, previously identified as the major p130 phosphotyrosyl protein in src-transformed cells. Bombesin stimulated p130 tyrosine phosphorylation with kinetics and concentration dependence similar to those observed for p125FAK. This is the first report to identify substrates for neuropeptide-stimulated tyrosine phosphorylation; the finding that one of these substrates is a tyrosine kinase suggests the existence of a novel signal transduction pathway in the action of mitogenic neuropeptides.

3T3 Cells↗
PubMed1991

Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells.

Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells.

3T3 Cells↗
PubMed1991

Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells.

The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of Mr 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of 32Pi-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C gamma, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Mutant alpha subunits of Gi2 inhibit cyclic AMP accumulation.

One or more of three Gi proteins, Gi1-3, mediates hormonal inhibition of adenylyl cyclase. Whether this inhibition is mediated by the alpha or by the beta gamma subunits of Gi proteins is unclear. Mutations inhibiting the intrinsic GTPase activity of another G protein, the stimulatory regulator of adenylyl cyclase (Gs), constitutively activate it by replacing either of two conserved amino acids in its alpha subunit (alpha s). These mutations create the gsp oncogene which is found in human pituitary and thyroid tumours. In a second group of human endocrine tumours, somatic mutations in the alpha subunit of Gi2 replace a residue cognate to one of those affected by gsp mutations. This implies that the mutations convert the alpha i2 gene into a dominantly acting oncogene, called gip2, and that the mutant alpha i2 subunits are constitutively active. We have therefore assessed cyclic AMP accumulation in cultured cells which stably or transiently express exogenous wild-type alpha i2 complementary DNA or either of two mutant alpha i2 cDNAs. The results show that putatively oncogenic mutations in alpha i2 constitutively activate the protein's ability to inhibit cAMP accumulation.

Adenylate Cyclase Toxin↗
PubMed1990

Increased mitogenic responsiveness of Swiss 3T3 cells expressing constitutively active Gs alpha.

Mutational replacement of glutamine-227 with a leucine residue in the GTP-binding domain of the alpha subunit of GS (Q227L alpha S) reduces its ability to hydrolyse GTP and causes constitutive activation of the mutant protein. Expression in Swiss 3T3 fibroblasts of Q227L alpha S caused markedly increased basal adenylyl cyclase activity, enhanced intracellular cyclic AMP (cAMP) accumulation and increased mitogenic sensitivity in response to forskolin and the potent phosphodiesterase inhibitor Ro 20-1724. These results support a role for cAMP in the regulation of cell proliferation, and suggest that alterations in a G protein can directly modify the ability of cells to respond mitogenically to extracellular factors.

Adenylyl Cyclases↗
PubMed1988

Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking.

We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups.

Affinity Labels↗
PubMed1987

Inhibition of bombesin-induced mitogenesis by pertussis toxin: dissociation from phospholipase C pathway.

Prior incubation of quiescent cultures of Swiss 3T3 cells with pertussis toxin selectively inhibited the stimulation of DNA synthesis induced by peptides of the bombesin family. While pertussis toxin blocked mitogenesis at an early stage in the action of the peptide, the toxin did not impair the rapid stimulation of polyphosphoinositide breakdown, Ca2+ mobilization or activation of protein kinase C promoted by bombesin. Thus, inhibition of bombesin-induced mitogenesis by pertussis toxin can be dissociated from inactivation of the phospholipase C signalling pathway.

Animals↗
PubMed1987

Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking.

The cross-linking agent ethylene glycol-bis(succinimidyl succinate) was used to covalently link 125I-labeled gastrin releasing peptide (125I-GRP) to an Mr 75,000-85,000 surface protein in Swiss 3T3 cells that displays many characteristics of a specific receptor for peptides of the bombesin family. This protein was not present in other cell lines which do not exhibit receptors for bombesin-like peptides. Unlabeled GRP competed for affinity labeling of the Mr 75,000-85,000 protein in a concentration-dependent manner, and other bombesin-related peptides also inhibited the cross-linking of 125I-GRP to this component. In contrast, high concentrations of a variety of other peptide hormones and mitogens had no effect. Affinity labeling of the Mr 75,000-85,000 protein was dependent on the concentration of 125I-GRP and exhibited saturability. 125I-GRP affinity labeling of this protein was also demonstrated by two-dimensional gel electrophoresis. These studies suggest that an Mr 75,000-85,000 surface protein with an isoelectric point of 6.0 to 6.5 is a major component of the receptor for peptides of the bombesin family in Swiss 3T3 cells.

Affinity Labels↗