Biomedical subjects

K Yokote

Publications and source records attributed to K Yokote.

At least 19 recordsLinked to original sources

PubMed1999

Enhanced expression of osteopontin by high glucose in cultured rat aortic smooth muscle cells.

Atherosclerotic vascular disease is a major complication of diabetic patients, and osteopontin has recently been implicated in the development of atherosclerosis. In the present study, we have investigated the effects of high glucose on expression of osteopontin in cultured rat aortic smooth muscle cells. High concentrations of glucose increased osteopontin secretion from the cells, and the increased secretion was completely inhibited by an inhibitor of protein kinase C, GF109203X. Northern blot analysis confirmed the enhanced effect of glucose on expression of osteopontin mRNA. Promoter activity of osteopontin, measured using the osteopontin promoter/luciferase expression vector system, was increased by high glucose, and the enhanced effect was completely inhibited by GF109203X. Glucosamine also increased the promoter activity of osteopontin, and azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (the key enzyme of the hexosamine pathway), profoundly inhibited high glucose-mediated increase in the promoter activity. Taken together, these data indicate that high glucose enhances the expression of osteopontin at the transcriptional level possibly through the activation of protein kinase C as well as the hexosamine pathway. Our results suggest that osteopontin could play a role in the development of diabetic vascular complications.

Animals↗

PubMed1999

Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration.

Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-gamma, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation.

Actins↗

PubMed1998

Fibronectin stimulates transcription of the platelet-derived growth factor beta-receptor in cultured rat aortic smooth muscle cells.

Fibronectin seems to play an important role in promoting the characteristic changes of vascular smooth muscle cells in diabetes mellitus including overexpression of the platelet-derived growth factor beta-receptor. To determine the regulatory mechanism of the beta-receptor by fibronectin, we have analyzed the effect of fibronectin on the expression of the beta-receptor in cultured rat aortic smooth muscle cells using the beta-receptor promoter/luciferase expression vector system. Fibronectin was found to stimulate the expression of the beta-receptor at the transcriptional level. Both a MEK1 inhibitor PD98059 and a tyrosine kinase inhibitor herbimycin A significantly inhibited the fibronectin-stimulated receptor transcription. Herbimycin A also completely inhibited the fibronectin-stimulated increase in tyrosine phosphorylation of focal adhesion kinase. These data suggest the involvement of the integrin-mediated mitogen-activated protein kinase pathway downstream of fibronectin stimulation in the activation process of the beta-receptor promoter.

Animals↗

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

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

Administration of a small amount of lard enhances intimal thickening in the balloon catheter injury model without affecting serum lipids.

Effects of fatty acids on intimal thickening induced by a balloon catheter injury model were investigated by feeding rabbits a small amount of either lard [L] or fish oil [F]. Serum lipids of these groups were not different from those of basal diet-fed rabbits [controls] after 4 weeks of feeding. Serum saturated fatty acids such as 14:0, 16:0, and 18.0 were significantly greater in the L-fed rabbits compared with controls, but those of the aorta were not significantly different. Fatty acid composition of the F-fed rabbits was only different from that of the controls in that n-3 fatty acids slightly increased. The mean and maximum intimal thickening 2 weeks after ballooning, carried out 2 weeks after feeding, were significantly higher in the carotid arteries of the L-fed rabbits than in the controls. The intimal thickening of the F-fed rabbits did not significantly differ from that of the controls. These results suggest that lard promotes the formation of the smooth muscle cell dominant type of arteriosclerosis without affecting serum lipid levels.

Animal Feed↗

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

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

Identification of an ubiquitin-ligation system for the epidermal-growth-factor receptor--herbimycin A induces in vitro ubiquitination in rabbit-reticulocyte lysate.

Some receptor tyrosine kinases such as the receptors for epidermal-growth factor (EGF) and platelet-derived growth factor undergo polyubiquitination as a consequence of ligand binding. The EGF receptor is also ubiquitinated by treatment with herbimycin A, an ansamycin antibiotic widely used as a tyrosine kinase inhibitor. To investigate the mechanism of the receptor ubiquitination, we have established an assay system in which herbimycin-A-induced ubiquitination processes can be analyzed in vitro. We now show that herbimycin A treatment of the purified EGF receptor induces polyubiquitination of the receptor in rabbit-reticulocyte lysate. Both DEAE unadsorbed material (fraction I) and high salt eluate (fraction II) of the reticulocyte lysate are involved cooperatively in the ubiquitination process, where the ubiquitin-conjugating enzyme UBC4 can functionally substitute for fraction I. A ubiquitin-protein ligase-like activity, partially purified from fraction II by DEAE anion-exchange chromatography, also functions in concert with UBC4. The precise mechanism of herbimycin A-induced ubiquitination of the EGF receptor is not fully understood, however, our present findings suggest that direct interaction with herbimycin A results in some modification of the receptor which is recognized by the ubiquitin-conjugating system in rabbit-reticulocyte lysate.

Animals↗

PubMed1997

Cultured retinal pericytes stimulate in vitro angiogenesis of endothelial cells through secretion of a fibroblast growth factor-like molecule.

Interaction between cultured endothelial cells (EC) and pericytes (PC) was studied in vitro to clarify the mechanism of diabetic proliferative retinopathy. Conditioned medium (CM) from retinal PC strongly increased the proliferation and moderately stimulated migration of retinal EC. Moreover, CM from PC caused stimulation of angiogenesis of retinal EC and umbilical cord vein EC in vitro at the same extent as basic fibroblast growth factor (bFGF). PC also stimulated angiogenesis by EC in mixed cultures. The angiogenic, proliferative and migration activities in CM from PC were inhibited by an antibody to bFGF. These data suggest that PC play an important role in angiogenesis through secretion of an FGF-like molecule.

Animals↗

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

Phosphatidylinositol 3-kinase activity is required for the activation process of focal adhesion kinase by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is one of the agents which stimulate increase in phosphotyrosine content of focal adhesion kinase (FAK) in cultured cells. In the present study we report that wortmannin, a highly specific and potent inhibitor of the catalytic subunit of mammalian phosphatidylinositol (PI) 3-kinase, completely abolishes PDGF-BB-mediated increase in tyrosine phosphorylation of FAK in human umbilical vein smooth muscle cells. Furthermore, analysis of the wild-type and mutant human PDGF beta-receptors stably expressed in porcine aortic endothelial cells also demonstrates that the Y740/751F mutant receptor, which cannot interact with PI 3-kinase due to the mutational alteration of its binding sites for PI 3-kinase, fails to increase FAK phosphorylation after PDGF-BB stimulation. These data suggest the requirement for PI 3-kinase activity in the activation process of FAK downstream of the PDGF receptor.

Androstadienes↗

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

[Indication and method of chest wall reconstruction].

Fifty two patients with chest wall resection were reviewed, with emphasis upon 16 patient with chest wall reconstruction. The latter 16 patient consisted of 6 with metastatic tumor, 3 of primary lung cancer, 2 of breast cancer, one of primary chest wall tumor, and others. Before 1985, reconstruction after chest wall resection was conducted in four cases by using methyl methacrylate (Resin). One patient developed erosion of the overlying skin due to protrusion of the edge of Resin-plate with delayed wound healing. Since 1986, we have employed muscle or myocutaneous flap and/or Marlex mesh in reconstruction of the chest wall defect. Twelve patients underwent surgery in this way. Neither paradoxical movement of the chest wall nor respiratory distress developed in the postoperative course of any patient. Thirty six of fifty two patients underwent chest wall resection without following reconstruction as in the former group. Of them one patient of anterior chest wall resection developed respiratory failure. We conclude that rib resection involving as many as three or more in the anterior chest wall, or four rib resection or more in the lateral chest wall, if the area of the defect is greater than 100 cm2, chest wall reconstruction is indicated. Moreover, we believe that muscle or myocutaneous flap and/or Marlex mesh in the best way of reconstruct in following chest wall resection.

Biocompatible Materials↗

PubMed1995

PDGF-BB triggered cytoplasmic calcium responses in cells with endogenous or stably transfected PDGF beta-receptors.

Platelet-derived growth factor-BB (PDGF-BB) triggered signal transduction was investigated in human foreskin fibroblasts with endogenous PDGF beta-receptors, and porcine aortic endothelial (PAE) cells with stably transfected PDGF beta-receptors. Immunoprecipitation and immunoblotting showed that PDGF induced dose-dependent autophosphorylation of PDGF beta-receptor, and the PLC-gamma associates with autophosphorylated PDGF beta-receptors and becomes phosphorylated. Activation of PLC-gamma is known to induce fluctuations of the concentration of cytoplasmic calcium ([Ca2+]i). Microfluorometry and digital imaging were employed for measurements of the concentration of [Ca2+]i. In both cell types the growth factor induced four types of [Ca2+]i responses; no rise, a small and sluggish monophasic rise, a biphasic rise with an initial transient peak followed by a sustain elevation, and finally regular oscillations. The frequencies and amplitudes of the oscillatory responses were independent of agonist concentration after stimulation with PDGF-BB. Latency, the period from application of stimulus to the first [Ca2+]i peak, was reduced at higher concentrations of agonist. Also, the proportion of responding cells increased with higher concentrations of ligand. Oscillations of [Ca2+]i were elicited at submaximal concentrations of agonist. In PAE cells PDGF-BB triggered a single [Ca2+]i peak in absence of external Ca2+. Ligand-induced oscillations and sustained increases of [Ca2+]i were counteracted by the inorganic Ca2+ channel blocker Ce3+. These results show that similar types of [Ca2+]i responses occur in different cell types independently of whether the PDGF beta-receptors are expressed endogeneously or after transfection. Potentially, the different [Ca2+]i responses have distinct physiological consequences.

Animals↗

PubMed1995

Bilateral intralobar pulmonary sequestration.

A 34-year-old male presented with an infected intralobar pulmonary sequestration of the left lower lobe. Aortography revealed bilateral anomalous systemic arteries, originating in the lower level of the descending thoracic aorta, to the lower lobe on each side. The portion of the right lower lobe, which was perfused by the anomalous systemic artery was seen otherwise normal in anatomy without any recognizable sequestered lung tissue. The patient underwent a left postero-lateral thoracotomy on June 22, 1994. Each aberrant artery was recognized to take off from a common branch of the descending aorta at the level of the diaphragm. A left lower lobectomy with division of the left aberrant artery as well as ligation of the right anomalous artery were done. A postoperative pulmonary perfusion scan depicted normal uptake of radioactivity in the right lower lobe, suggesting normal pulmonary arterial perfusion to the area receiving previously the anomalous systemic arterial flow. An anomalous systemic artery perfusing an otherwise normal lung can be classified as one of the forms of intralobar pulmonary sequestration and could be ligated without resection of the involved area of the lung.

Adult↗

PubMed1995

[Surgical treatment of posterior mediastinal teratoma].

A 22-year-old male patient was admitted to our hospital because of an abnormal shadow on a chest X-ray taken for routine examination. Preoperative diagnostic work up including chest computed tomography and magnetic resonance imaging suggested that the tumor would most likely to be a posterior mediastinal lipoma. Through a right thoracotomy the tumor was removed successfully. Histologically it proved to be a mature teratoma. The incidence, etiology and diagnostic procedure of the mediastinal teratoma was discussed.

Adult↗

PubMed1994

Role of phospholipase A2 in expression of the scavenger pathway in cultured aortic smooth muscle cells stimulated with phorbol 12-myristate 13-acetate.

We have demonstrated that cultured intimal smooth muscle cells (SMC) from thickened intima can metabolize acetylated low-density lipoprotein (LDL) by a scavenger pathway, but medial SMC from normal arteries cannot. In this study we investigated the expression mechanism of the scavenger pathway in medial SMC using a phorbol ester. Medial SMC were incubated with 10(-10)-10(-7) M phorbol 12-myristate 13-acetate (PMA) for 1-24 h and then their degradation of 125I-labelled acetylated LDL was assayed. Unstimulated SMC degraded little acetylated LDL, but incubation for 24 h with PMA dose-dependently stimulated its degradation by SMC, the optimal PMA concentration being 1 x 10(-8) M. Induction of expression of the scavenger pathway required more than 4 h of incubation with PMA and was completely inhibited by cycloheximide. In addition expression of the scavenger pathway was not transient but stable. Induction of expression of the scavenger pathway by PMA was not inhibited by protein kinase C inhibitors, but was inhibited about 50% by phospholipase A2 inhibitors. The study, using various phorbol esters, indicated that induction of the scavenger pathway was well correlated with their ability to stimulate phospholipase A2 in medial SMC but not with their ability to activate protein kinase C. Moreover, incubation with exogenous phospholipase A2 (0.1-10 units/ml) or its product, lysophosphatidylcholine (0.01-100 micrograms/ml) dose-dependently increased degradation of 125I-labelled acetylated LDL in medial SMC. Lysophosphatidylcholine was most effective in various lysophospholipids. These results suggest that PMA induced the scavenger pathway in part by stimulating phospholipase A2 in medial SMC, and that a product, lysophosphatidylcholine, is a mediator of expression of the scavenger pathway.

Animals↗