Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae.

The Rho small GTP-binding protein family regulates various actomyosin-dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small-budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.

Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae.

The small GTP-binding proteins of the Rho family, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, cell motility and cytokinesis, through reorganization of actin cytoskeleton. Rho GDI is a general regulator which forms a complex with the GDP-bound inactive form of the Rho family members and inhibits their activation. We have purified Rho GDI from the yeast Saccharomyces cerevisiae, cloned its gene, and named it RDII (Rho GD). In this study, we have further characterized yeast Rho GDI. Rho GDI was found in the cytosol by immunoblot and immunofluorescence microscopic analyses. Rho1p and Cdc42p were co-immunoprecipitated with Rho GDI from the cytosol. This immunoprecipitated Rho1p was mainly bound to GDP. In the disruption mutant of Rho GDI, which did not show any apparent phenotype, both Rho1p and Cdc42p were also present in the cytosol. These results indicate that yeast Rho GDI possesses properties similar to those of mammalian Rho GDI, and that there is a cytosolic factor which functionally substitutes for Rho GDI in yeast.

Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility.

Hepatocyte growth factor (HGF) induced motility of cultured mouse keratinocytes (308R cells). This HGF-induced cell motility was inhibited by microinjection of either rho GDI, an inhibitory GDP/GTP exchange protein for rho p21 small GTP-binding protein, or a botulinum exoenzyme C3 which is known to selectively impair the function of rho p21 by ADP-ribosylating its effector domain. The rho GDI action was prevented by comicroinjection with the guanosine 5'-(3-0-thio)triphosphate (GTP gamma S)-bound active form of rhoA p21, and the C3 action was prevented by comicroinjection with a rhoA p21 mutant (rhoAIle41 p21) which is resistant to the C3 action. The HGF-induced cell motility was not inhibited by microinjection of a dominant negative rac1 p21 mutant (rac1Asn17 p21) or a dominant negative Ki-ras p21 mutant (Ki-rasAsn17 p21). Microinjection of the GTP gamma S-bound form of rac1 p21 or a dominant active Ki-ras p21 mutant (Ki-rasVal12 p21) did not induce cell motility. These results indicate that both rho p21 and rho GDI, but neither rac p21 nor ras p21, are involved in the HGF-induced cell motility. However, microinjection of the GTP gamma S-bound form of rhoA p21 alone did not induce cell motility in the absence of HGF, suggesting that activation of rho p21 is necessary but not sufficient for the HGF-induced cell motility. The HGF-induced cell motility was mimicked by 12-0-tetradecanoyl-phorbol-13-acetate, a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore. The phorbol ester-induced cell motility was also inhibited by microinjection of rho GDI or C3. These results indicate that both rho p21 and rho GDI are also involved in the phorbol ester-induced cell motility.

Stimulatory interaction between vascular endothelial growth factor and endothelin-1 on each gene expression.

The precise regulation of cell growth in the vascular wall maintains vascular integrity, and its disruption leads to cardiovascular disorders including atherosclerosis and restenosis. Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells, and endothelin-1 (ET-1) is known to stimulate the proliferation of smooth muscle cells. The aim of this study was to explore a potential interaction between VEGF and ET-1 on each expression in vascular cells. VEGF enhanced preproET-1 mRNA expression and ET-1 secretion in bovine aortic endothelial cells (BAECs). Similarly, in rat vascular smooth muscle cells (VSMCs), ET-1 enhanced VEGF mRNA expression and stimulated VEGF secretion. ET-1-induced VEGF mRNA expression was abolished by a selective ET(A) receptor antagonist, BQ-485, but not by an ET(B)-selective blocker, BQ-788. It was also inhibited by pretreatment with actinomycin D but not by pretreatment with cycloheximide. Furthermore, the actinomycin D chase experiment revealed that ET-1 did not alter VEGF mRNA stability. Coculture of BAECs and VSMCs enhanced both ET-1 and VEGF gene expression in these cells, and the conditioned media from BAECs and VSMCs reproduced the augmentation of each gene expression, which was partially inhibited by BQ-485 or an antibody specific to VEGF. Our results indicate that VEGF and ET-1 have stimulatory interactions on each expression, which may play an important role in concomitant proliferation of endothelial and smooth muscle cells in the vascular wall.

Vascular endothelial growth factor increases endothelin-converting enzyme expression in vascular endothelial cells.

Endothelin-converting enzyme-1 (ECE-1) is a key enzyme in endothelin processing. Although it has been revealed that ECE-1 expression increases in vascular wall after balloon injury in vivo experimental models, the regulation of ECE-1 expression in vitro remains undefined. In this study, we demonstrated that the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF) increased ECE-1 expression in cultured bovine aortic endothelial cells (BAEC). Northern blot analysis revealed that VEGF increased ECE-1 mRNA expression in 12-24 hours by 2-fold in BAEC, and this effect was dose-dependent. VEGF also increased ECE-1 protein expression detected by immunoblotting in 36 hours in BAEC. Therefore, VEGF increased ECE-1 expression in BAEC, which suggests that ECE-1 induction by VEGF may be involved in endothelin-system upregulation under pathological conditions such as neointimal formation and atherosclerosis.

Molecular cloning and characterization of yeast rho GDP dissociation inhibitor.

We have previously isolated rho GDP dissociation inhibitor (rho DGI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids. rho GDI inhibits the GDP/GTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.

Stretch force on vascular smooth muscle cells enhances oxidation of LDL via superoxide production.

Hemodynamic forces on vasculature profoundly influence atherogenesis. We examined the effect of stretch force on the oxidation of low-density lipoprotein (LDL) by rat aortic smooth muscle cells (RASM) and superoxide production. Stretch force was imposed on RASM cultured on deformable dishes by stretching the dishes. Incubation of native LDL with static RASM for 24 h resulted in LDL oxidation as indicated by increases in thiobarbituric acid-reacting substances from 9.5 +/- 2.3 to 24.5 +/- 2.3 nmol malondialdehyde/mg. Stretch force on RASM augmented cell-mediated LDL oxidation to 149.3 +/- 17.1% concomitantly with increase in superoxide production. LDL oxidation was inhibited by superoxide dismutase or depletion of the metal ion in the culture medium, indicating that it was a metal ion-dependent and superoxide-mediated process. The enhancement of LDL oxidation by stretch force was inhibited by diphenyliodonium, indicating the involvement of the NADH/NADPH oxidase system. Our findings suggest that the increased oxidant stress induced by stretch force is one of the potential mechanisms whereby hypertension facilitates atherosclerosis.

Transforming growth factor-beta1 and protein kinase C synergistically activate the c-fos serum response element in myocardial cells.

We previously reported that transforming growth factor-beta1 (TGF-beta1) potentiated alpha1-adrenergic and stretch-induced c-fos mRNA expression and norepinephrine (NE)-induced amino acid incorporation in rat cultured myocardial cells (MCs). In the present study, we attempted to explore the mode of TGF-beta1 action for c-fos gene expression in MCs. In the transient transfection assay, TGF-beta1 potentiated NE- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated c-fos promoter/enhancer, but not forskolin-activated c-fos promoter/enhancer. The c-fos serum response element (SRE) and the TPA response element (TRE) were responsible for TGF-beta1-induced potentiation of the NE or TPA action. Although TGF-beta1 activated not only the wild-type c-fos SRE, but also the mutated c-fos SRE, which contains an intact binding site for the serum response factor (SRF) but lacks the ternary complex factor (TCF) binding site, TPA activated the wild-type c-fos SRE but not the mutated c-fos SRE. TGF-beta1 did not potentiate the effects of TPA on the activation of mitogen-activated protein kinase (MAPK) and the phosphorylation of Elk-1 and SAP-1a, which belong to TCF at the c-fos SRE. These results indicate that TGF-betaf potentiates the c-fos SRE activated by PKC through the SRF binding site. TGF-beta1 is involved in the regulation of c-fos gene expression through the c-fos SRE and is subsequently involved in the regulation of the gene which has the TRE in the promoter/enhancer region.