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No abstract available.

Cell motility is essential for a wide range of cellular activities including anigogenesis as well as metastasis of tumor cells. Ras has been implicated in cell migration and invasion, and functions at upstream of mitogen-activated protein kinase (MAPK) families, which include extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In the present study, we examined the role of JNK in endothelial cell motility using stable transfectant (DAR-ECV) of ECV304 endothelial cells expressing previously established oncogenic H-Ras (leu 61). DAR-ECV cells showed an enhanced angiogenic potential and motility (approximately 2-fold) compared to ECV304 cells. Western blot analysis revealed constitutive activation of JNK in DAR-ECV cells. Pretreatment of JNK specific inhibitors, curcumin and all trans-retinoic acid, decreased the basal motility of DAR-ECV cells in a dose-dependent manner. These inhibitors also suppressed the motility stimulated by known JNK agonists such as TNFalpha and anisomycin. To further confirm the role of JNK, ECV304 cells expressing dominant active SEK1 (DAS-ECV) were generated. Basal non-stimulated levels of the cellular migration were greater in DAS-ECV clones than those in control ECV304 cells. These results suggest that Ras-SEK1-JNK pathway regulates motility of endothelial cells during angiogenesis.
Anisomycin/pharmacology ; Cell Line ; *Cell Movement ; Curcumin/pharmacology ; Endothelium, Vascular/cytology/*physiology ; Enzyme Activation ; Enzyme Inhibitors/pharmacology ; Extracellular Matrix/metabolism ; Genes, ras/genetics ; Human ; Matrix Metalloproteinases/physiology ; Mitogen-Activated Protein Kinases/*metabolism ; Neovascularization, Physiologic ; Support, Non-U.S. Gov't ; Tretinoin/pharmacology ; Tumor Necrosis Factor/pharmacology ; Umbilical Veins/cytology ; Urinary Plasminogen Activator/physiology

Cell motility is essential for a wide range of cellular activities including anigogenesis as well as metastasis of tumor cells. Ras has been implicated in cell migration and invasion, and functions at upstream of mitogen-activated protein kinase (MAPK) families, which include extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In the present study, we examined the role of JNK in endothelial cell motility using stable transfectant (DAR-ECV) of ECV304 endothelial cells expressing previously established oncogenic H-Ras (leu 61). DAR-ECV cells showed an enhanced angiogenic potential and motility (approximately 2-fold) compared to ECV304 cells. Western blot analysis revealed constitutive activation of JNK in DAR-ECV cells. Pretreatment of JNK specific inhibitors, curcumin and all trans-retinoic acid, decreased the basal motility of DAR-ECV cells in a dose-dependent manner. These inhibitors also suppressed the motility stimulated by known JNK agonists such as TNFalpha and anisomycin. To further confirm the role of JNK, ECV304 cells expressing dominant active SEK1 (DAS-ECV) were generated. Basal non-stimulated levels of the cellular migration were greater in DAS-ECV clones than those in control ECV304 cells. These results suggest that Ras-SEK1-JNK pathway regulates motility of endothelial cells during angiogenesis.
Anisomycin/pharmacology ; Cell Line ; *Cell Movement ; Curcumin/pharmacology ; Endothelium, Vascular/cytology/*physiology ; Enzyme Activation ; Enzyme Inhibitors/pharmacology ; Extracellular Matrix/metabolism ; Genes, ras/genetics ; Human ; Matrix Metalloproteinases/physiology ; Mitogen-Activated Protein Kinases/*metabolism ; Neovascularization, Physiologic ; Support, Non-U.S. Gov't ; Tretinoin/pharmacology ; Tumor Necrosis Factor/pharmacology ; Umbilical Veins/cytology ; Urinary Plasminogen Activator/physiology

Angiogenesis is a fundamental biological process including endothelial cell adhesion, migration, invasion and tube formation. Integrin receptors of endothelial cells play important roles in angiogenesis. They mediate cell-cell contact and cell adhesion to extracellular matrix. Roles of integrins have been described for a number of cell types. ECV304 endothelial cells were known to overexpress alpha3beta1 integrin and to form tube like structure in 3-D Matrigel culture. However the function of alpha3beta1 integrin in endothelial cells remains to be determined. Therefore, we have investigated morphological characteristics of ECV304 cells and roles of alpha3beta1 integrin in angiogenesis. To elucidate several characteristics, ECV304 endothelial cells were compared with HUVEC in the aspect of morphology, localization of integrins, angiogenesis pattern. In addition, role of alpha3beta1 integrin were analyzed in the aspect of endothelial cell binding, migration, invasion and tube formation on Matrigel. The result showed that alpha3beta1 integrin overexpressed ECV304 endothelial cells showed strong adhesiveness to extracellular matrix proteins, and high migration and invasion activities. Furthermore, expression of alpha3beta1 integrin was increased according to time course during in vitro culture and was continuously strong in ECV304 cells on 3-D Matrigel culture. These results indicate that alpha3beta1 integrin is able to be a critical component in control of angiogenesis by regulation of cell adhesion, migration, invasion and tube formation of ECV304 endothelial cells.
Adhesiveness ; Biological Processes ; Cell Adhesion ; Endothelial Cells* ; Extracellular Matrix ; Extracellular Matrix Proteins ; Integrin alpha3beta1* ; Integrins

Neuronal differentiation is a complex biological process accompanying cytoskeletal reorganization, including neurite outgrowth and growth cone formation. Therefore, neuronal differentiation is critically regulated by actin-related signaling proteins, such as small Rho GTPases, guanine nucleotide exchange factors (GEFs), and myosins. This study will demonstrate the change in activity of three small Rho GTPases, Rac, Cdc42, and Rho A, by treatment with blebbistatin (BBS), a specific inhibitor for myosin, during bFGF-induced neurite outgrowth in PC12 cells. Treatment with BBS induced morphological changes in growth cones and neurites during differentiation. A marked increase in protrusion and filopodia structures in growth cones, the shaft of neuritis, and cell membranes was observed in the cells treated with BBS. Activity of Rho GTPases showed the alterations in response to BBS. Activities of both Rac and Rho A were inhibited by BBS in a time-dependent manner. By contrast, Cdc42 activity was not changed by BBS. These results suggest that inactivation of myosin II by BBS induced morphological changes in neurites and growth cones and distinct regulation of three Rho GTPases during differentiation of PC12 cells.
Animals ; Biological Processes ; Cell Membrane ; Growth Cones ; Guanine Nucleotide Exchange Factors ; Heterocyclic Compounds with 4 or More Rings ; Myosin Type II ; Myosins ; Neurites ; Neuritis ; Neurons ; PC12 Cells* ; Proteins ; Pseudopodia ; rho GTP-Binding Proteins*

BACKGROUND: Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. METHODS: Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. RESULTS: Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34⁺CD43⁺ hematopoietic progenitor cells (HPCs) and CD34⁺CD45⁺ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. CONCLUSION: In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.
Animals ; Cytokines ; Embryonic Stem Cells ; Hematopoietic Stem Cells* ; Human Embryonic Stem Cells* ; Humans* ; In Vitro Techniques ; Methods* ; Pluripotent Stem Cells ; Stem Cells

Amphiphysin I and II, proteins enriched in nerve terminals, form heterodimers and interact with dynamin and synaptojanin through their Src homology 3 (SH3) domain. In order to study the expression profile of Amphs in cells and tissues and the interaction state with other cellular molecules, we have prepared specific monoclonal antibodies (mAbs) designed to bait N-terminus, middle part, and C-terminus domains of Amph I, respectively by immunizing with the expressed smaller domain molecules using the GST gene fusion system. The expression of Amphs was found to be most abundant in PC12 cells, followed by B103 cells and vascular smooth muscle cells. Western blot analysis showed a relatively high level expression of Amphs that were found in both mouse and rat brain. There appeared to be some species difference in the expression pattern, i.e. Amphs are present more in the testis than in the lungs in rats, however, they are reversed in mice. Characterization of the mAbs revealed that clone 14-23 precipitated Amph I and II, whereas clone 8-2 could only precipitate Amph I. In addition, clathrin and dynamin in a complex with Amph were captured in the precipitate formed by mAbs and identified by the Western blot analysis. Cellular distribution of Amph was visualized with confocal immunofluorescence microscopy performed using the labeled-mAbs. Taken together, these results demonstrated that mAbs provided an excellent measure for studying Amphs' expression profile and their interacting proteins.
Animal ; *Antibodies, Monoclonal ; Blotting, Western ; Brain/metabolism ; Cells, Cultured ; Dimerization ; Enzyme-Linked Immunosorbent Assay ; Glutathione Transferase/metabolism ; Human ; Mice ; Mice, Inbred BALB C ; Microscopy, Confocal ; Nerve Tissue Proteins/*chemistry/*immunology ; PC12 Cells ; Precipitin Tests ; Protein Binding ; Protein Structure, Tertiary ; Rats ; Recombinant Fusion Proteins/metabolism ; Support, Non-U.S. Gov't ; src Homology Domains

Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a betaPIX GEF-dependent manner. betaPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of betaPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of betaPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of betaPIX-Rac1. Taken together, our results suggest that betaPIX is involved in Ang II-induced VSMC migration.
1-Phosphatidylinositol 3-Kinase/metabolism ; Angiotensin II/*metabolism ; Animals ; *Cell Movement ; Cells, Cultured ; Guanine Nucleotide Exchange Factors/genetics/*metabolism ; Muscle, Smooth, Vascular/cytology ; Myocytes, Smooth Muscle/*cytology ; NADPH Oxidase/metabolism ; Protein Kinase C/metabolism ; RNA, Small Interfering/genetics ; Rats ; Rats, Sprague-Dawley ; p21-Activated Kinases/metabolism ; rac1 GTP-Binding Protein/metabolism ; src-Family Kinases/metabolism

Glutamate induced rapid phosphorylation of moesin, one of ERM family proteins involved in the ligation of membrane to actin cytoskeleton, in rat hippocampal cells (JBC, 277:16576-16584, 2002). However, the identity of glutamate receptor has not been explored. Here we show that a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is responsible for glutamate-induced RhoA activation and phosphorylation of moesin. Glutamate induced phosphorylation at Thr-558 of moesin was still detectible upon chelation of Ca(2+), suggesting involvement of AMPA receptor instead of N-methyl D-Aspartate (NMDA) receptor in this phosphorylation of moesin. AMPA but not NMDA- induced moesin phosphorylation was independent of Ca(2+). Both AMPA and NMDA but not Kainate induced moesin phosphorylation at similar levels. However, the kinetics of phosphorylation varied greatly between AMPA and NMDA where AMPA treatment rapidly increased phosphomoesin, which reached a maximum at 10 min after treatment and returned to a basal level at 30 min. In contrast, NMDA-induced phosphorylation of moesin reached a maximum at 30 min after treatment and was remained at higher levels at 60 min. A possible involvement of RhoA and its downstream effector, Rho kinase in the AMPA receptor-triggered phosphorylation of moesin was also explored. The kinetics for the glutamate- induced membrane translocation of RhoA was similar to that of moesin phosphorylation induced by AMPA. Moreover, Y-27632, a specific Rho kinase inhibitor, completely blocked AMPA-induced moesin phosphorylation but had no effect on NMDA-induced moesin phosphorylation. These results suggest that glutamate-induced phosphorylation of moesin may be mediated through the AMPA receptor/RhoA/Rho kinase pathway.
Animals ; Calcium/metabolism ; Cell Line ; Excitatory Amino Acid Agonists/*metabolism ; Glutamic Acid/metabolism ; Kainic Acid/metabolism ; Microfilament Proteins/*metabolism ; N-Methylaspartate/*metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/metabolism ; Rats ; Receptors, AMPA/metabolism ; Receptors, N-Methyl-D-Aspartate/metabolism ; Research Support, Non-U.S. Gov't ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/*metabolism ; rhoA GTP-Binding Protein/*metabolism