Biomedical subjects

Yoshihiro Akimoto

Publications and source records attributed to Yoshihiro Akimoto.

At least 19 recordsLinked to original sources

PubMed2006

Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki rats.

Many nuclear and cytoplasmic proteins are O-glycosylated on serine or threonine residues with the monosaccharide beta-N-acetylglucosamine, which is then termed O-linked N-acetylglucosamine (O-GlcNAc). It has been shown that abnormal O-GlcNAc modification (O-GlcNAcylation) of proteins is one of the causes of insulin resistance and diabetic complications. In this study, in order to examine the relationship between O-GlcNAcylation of proteins and glucose-stimulated insulin secretion in noninsulin-dependent type (type 2) diabetes, we investigated the level of O-GlcNAcylation of proteins, especially that of PDX-1, and the expression of O-GlcNAc transferase in Goto-Kakizaki (GK) rats, which are an animal model of type-2 diabetes. By immunoblot and immunohistochemical analyses, the expression of O-GlcNAc transferase protein and O-GlcNAc-modified proteins in whole pancreas and islets of Langerhans of 15-week-old diabetic GK rats and nondiabetic Wistar rats was examined. The expression of O-GlcNAc transferase at the protein level and O-GlcNAc transferase activity were increased significantly in the diabetic pancreas and islets. The diabetic pancreas and islets also showed an increase in total cellular O-GlcNAc-modified proteins. O-GlcNAcylation of PDX-1 was also increased. In the diabetic GK rats, significant increases in the immunoreactivities of both O-GlcNAc and O-GlcNAc transferase were observed. PUGNAc, an inhibitor of O-GlcNAcase, induced an elevation of O-GlcNAc level and a decrease of glucose-stimulated insulin secretion in isolated islets. These results indicate that elevation of the O-GlcNAcylation of proteins leads to deterioration of insulin secretion in the pancreas of diabetic GK rats, further providing evidence for the role of O-GlcNAc in the insulin secretion.

Acetylglucosamine↗

PubMed2005

Toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an interface chip.

This paper reports a thermal lens microscope (TLM) detection coupled with capillary electrophoresis (CE) by using an interface chip (IFChip) to achieve highly sensitive detection with high reproducibility. Fused silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel on the IFChip. In comparison with an on-capillary detection method in CE-TLM, ca. 10-fold improvements in the reproducibility for peak height were obtained by using IFChips. The detection limit of an azo dye was estimated to be 3.6 x 10(-7)M (100 ppb), which was above 100-times lower than that of conventional absorbance detection. Toward further improvement of the detectability for nonfluorescent compounds, on-line sample preconcentration by sweeping was applied to the CE-TLM using the IFChip. Due to the sweeping effect, 3900000-fold increase in the sensitivity was successfully achieved.

Electrophoresis, Capillary↗

PubMed2005

ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.

Animals↗

PubMed2005

Tissue transglutaminase at embryo-maternal interface.

CONTEXT: Tissue transglutaminase (tTG) has a high affinity for fibronectin (FN) and is a coreceptor of both beta1 and beta3 integrin subunits. Considering the notion that FN and integrins have critical roles during the implantation process, this study was undertaken to elucidate the expression pattern and the potential physiological function of tTG at the embryo-maternal interface. METHODS: The primary cultures of human placentas from 15 legal elective abortions at the first trimester of normal pregnancies and endometrial biopsies of 12 female patients in the midluteal phase as well as normal trophoblastic cell lines (CRL) were employed to address these issues using several approaches, such as scanning and transmission electron microscopies, immunostaining for light and electron microscopies, western blotting, and function assays using GRGDSP hexapeptide and an antibody against tTG. RESULTS: The results demonstrated tTG expression on uterine pinopodes and lamellipodia of extravillous trophoblasts. The colocalization of tTG with beta1 and beta3 integrins and its interaction with alpha(v)beta3 integrin and integrin-associated proteins at focal adhesions of the extravillous trophoblasts were illustrated in the results of immunofluorescence, immunoblot, and coimmunoprecipitation studies. Furthermore, function assays revealed that tTG mediated the adhesion and spread of the placental cells on intact FN-coated and 42- and 110-kDa FN fragment-coated wells. CONCLUSION: In conclusion, our findings demonstrated for the first time that tTG actively participates in adhesion events at the embryo-maternal interface through its interaction with FN, at least in part, by activating integrin-signaling pathways.

Blotting, Western↗

PubMed2005

Mouse Na+/K+-ATPase beta1-subunit has a K+-dependent cell adhesion activity for beta-GlcNAc-terminating glycans.

A 48-kDa beta-N-acetylglucosamine (GlcNAc)-binding protein was isolated from mouse brain by GlcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse Na(+)/K(+)-ATPase beta1-subunit. When the recombinant FLAG-beta1-subunit expressed in Sf-9 cells was applied to a GlcNAc-agarose column, only the glycosylated 38- and 40-kDa proteins bound to the column. In the absence of KCl, little of the proteins bound to a GlcNAc-agarose column, but the 38- and 40-kDa proteins bound in the presence of KCl at concentrations above 1 mM. Immunohistochemical study showed that the beta1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-beta1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the Na(+)/K(+)-ATPase beta1-subunit is a potassium-dependent lectin that binds to GlcNAc-terminating oligosaccharides: it may be involved in neural cell interactions.

Acetylglucosamine↗

PubMed2005

O-GlcNAc modification of nucleocytoplasmic proteins and diabetes.

Nuclear and cytosolic proteins are glycosylated on serine or threonine residues by O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification is one of various posttranslational modifications and seems to be involved in the modulation of transcription and signal transduction. Accumulating data suggest a role for O-GlcNAc-modified proteins in diabetes, acting as a glucose sensor. It has been suggested that the hexosamine biosynthetic pathway is involved in the mechanism causing insulin resistance and diabetic complications. Excess glucose entering into the hexosamine biosynthetic pathway might cause elevated O-GlcNAc modification of various proteins. In this article, we review the current data regarding the relationship between O-GlcNAc modification and diabetes.

Acetylglucosamine↗

PubMed2005

Expression of Hex during feather bud development.

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.

Animals↗

PubMed2005

Involvement of Hex in the initiation of feather morphogenesis.

In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.

Animals↗

PubMed2004

Localization of HB9 homeodomain protein and characterization of its nuclear localization signal during chick embryonic skin development.

We detected HB9 protein during tarsometatarsal scale skin and late feather development. Immunofluorescent analyses with N-terminal 14 amino acids antiserum revealed that HB9 was strongly expressed in epidermal basal cells of the outer scale face in tarsometatarsal scale skin. Specific expression was also detected in dermal cells at the root region of the feather and around the feather follicle. Furthermore, we observed precise distribution of HB9 protein by immunoelectron microscopy. We detected HB9 protein not only in the nucleus, but also in the cytoplasm in tarsometatarsal scale skin. However, in feather skin HB9 protein was found in the nucleus but not in the cytoplasm. Cytoplasmic localization of HB9 protein in tarsometatarsal scale skin was observed especially in the endoplasmic reticulum and the Golgi apparatus. To address the mechanism of nuclear-cytoplasmic translocation, we determined the nuclear localization signal (NLS) sequences by using eukaryotic green fluorescent protein fusion protein in primary keratinocyte culture. Chick HB9 homeoprotein has two types of the NLS sequences in its homeodomain. One of them is a bipartite type as representatively found in Xenopus nucleoplasmin. The other is very similar to hexapeptide NLS sequences identified in pancreatic duodenum homeobox 1 (PDX1). These sequences functioned not only in keratinocytes but also in dermal fibroblasts. They are conserved in Xenopus, mouse, and human HB9 ortholog. These results indicate that HB9 protein might be involved in chick tarsometatarsal scale and feather development and that nuclear import of HB9 protein might be regulated by these NLS sequences in the homeodomain.

Amino Acid Sequence↗

PubMed2003

Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in rat cerebellar cortex.

O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous nucleocytoplasmic protein modification that has a complex interplay with phosphorylation on cytoskeletal proteins, signaling proteins and transcription factors. O-GlcNAc is essential for life at the single cell level, and much indirect evidence suggests it plays an important role in nerve cell biology and neurodegenerative disease. Here we show the localization of O-GlcNAc Transferase (OGTase) mRNA, OGTase protein, and O-GlcNAc-modified proteins in the rat cerebellar cortex. The sites of OGTase mRNA expression were determined by in situ hybridization histochemistry. Intense hybridization signals were present in neurons, especially in the Purkinje cells. Fluorescent-tagged antibody against OGTase stained almost all of the neurons with especially intense reactivity in Purkinje cells, within which the nucleus, perikaryon, and dendrites were most intensely stained. Using immuno-electron microscopic labeling, OGTase was seen to be enriched in euchromatin, in the cytoplasmic matrix, at the nerve terminal, and around microtubules in dendrites. In nerve terminals, immuno-gold labeling was observed around synaptic vesicles, with the enzyme more densely localized in the presynaptic terminals than in the postsynaptic ones. Using an antibody to O-GlcNAc, we found the sugar localizations reflected results seen for OGTase. Collectively, these data support hypothesized roles for O-GlcNAc in key processes of brain cells, including the regulation of transcription, synaptic vesicle secretion, transport, and signal transduction. Thus, by modulating the phosphorylation or protein associations of key regulatory and cytoskeletal proteins, O-GlcNAc is likely important to many functions of the cerebellum.

Acetylglucosamine↗

PubMed2003

Elevated expression of O-GlcNAc-modified proteins and O-GlcNAc transferase in corneas of diabetic Goto-Kakizaki rats.

PURPOSE: The hexosamine biosynthetic pathway is one of the possible mechanisms involved in diabetic keratopathy. The purpose of this study was to examine the role of O-glycoside-linked N-acetylglucosamine (O-GlcNAc) modification of proteins in the pathogenesis of diabetic keratopathy in the Goto-Kakizaki (GK) rat, which has spontaneous development of diabetes. METHODS: An anti-O-GlcNAc antibody, an anti-O-GlcNAc transferase antibody, and digoxigenin (DIG)-labeled cRNA probes were used to examine the localization of O-GlcNAc-modified proteins, O-GlcNAc transferase protein and mRNA in the corneas of diabetic GK rats and in those of nondiabetic Wistar rats. The corneas from Wistar rats were organ cultured in control medium or in medium containing 100 micro M O-(2-acetamide-2-deoxy-D-glucopyranosylidene) amino-N-phenyl-carbamate (PUGNAc), an inhibitor of O-GlcNAcase, the enzyme that removes O-GlcNAc from proteins. The morphologic changes were examined by electron microscopy. RESULTS: In normal corneas, immunoreactive O-GlcNAc and O-GlcNAc transferase were observed in the epithelial, endothelial, and stromal cells. In the diabetic corneas, their immunoreactivities in the epithelium were increased in intensity. Morphologically, the number of hemidesmosomes in the epithelial basal cells was lower than that in those cells from the nondiabetic rats. In some areas, the basement membrane had detached from the epithelial basal cells. The PUGNAc treatment of Wistar rat corneas increased the level of O-GlcNAc immunoreactivity and caused a decrease in the number of hemidesmosomes and the detachment of corneal epithelial cells from the basement membrane. CONCLUSIONS: The elevated expression of O-GlcNAc-modified proteins and O-GlcNAc transferase may play a causative role in the corneal epithelial disorders of diabetic GK rats.

Acetylglucosamine↗

PubMed2003

Alphavbeta3 integrin signaling pathway is involved in insulin-like growth factor I-stimulated human extravillous trophoblast cell migration.

IGF-I and -II provide paracrine and autocrine stimuli, respectively, for extravillous trophoblast (EVT) cell migration. This study examined the role of alpha(v)beta(3) integrin and its signaling pathway in IGF-I-stimulated migration. Migration assays were conducted using cultured EVT cells treated with or without IGF-I in the presence or absence of alphaIR3, Arg-Gly-Asp (RGD) hexapeptide, and antibody against alpha(v)beta(3) integrin. Morphological changes were studied using scanning electron microscopy. Colocalization of alpha(5)beta(1) alpha(v)beta(3) integrins, vinculin, focal adhesion kinase, and paxillin were determined by immuno-cytochemistry and immunoblotting. The results showed that IGF-I could stimulate EVT cell migration in a time- and dose-dependent manner and addition of alphaIR3, Arg-Gly-Asp hexapeptide, and antibody against alpha(v)beta(3) integrin attenuated the IGF-I migratory effect. Scanning electron microscopy images revealed that IGF-I promoted lamellipodia formation. Immunostaining and immunoblotting exhibited the colocalization of alpha(v)beta(3) integrin with phosphorylated focal adhesion kinase, paxillin, and vinculin at focal adhesions after IGF-I treatment. Immunoblotting demonstrated an increase in focal adhesion kinase and paxillin tyrosine phosphorylation followed by tyrosine phosphorylation of IGF-I receptor in a time- and dose-dependent manner. These findings indicated alpha(v)beta(3) integrin localization in the core of focal adhesions of EVT cells and that alpha(v)beta(3) integrin signaling pathways are activated in IGF-I-mediated migration of these cells.

Cell Movement↗

PubMed2003

Physiologic role of decidual beta1 integrin and focal adhesion kinase in embryonic implantation.

Implantation refers to a series of interactions between embryo and endometrium including hatching, attachment, and outgrowth. We investigated the expression and function of beta1 integrin and focal adhesion kinase (FAK) in human decidual cells during implantation. Immunofluorescent staining localized beta1 integrin to surfaces of cultured decidual cells. Double staining for beta1 integrin and mediators of intracellular signaling involving beta1 integrin, such as FAK and vinculin, colocalized beta1 integrin with these substances, suggesting that human decidual cells express beta1 integrin in the focal adhesion region. We next investigated the actions of beta1 integrin and FAK in implantation by co-culturing mouse embryos and human decidual cells. Mouse blastocysts attached to cultured decidual cells after embryo hatching, usually within 24 h of culture initiation. Blastocysts attached to decidual cells exhibited extensive outgrowth at 48 h. Treatment of decidual cells with an antibody against beta1 integrin or with an antisense FAK oligonucleotide did not affect hatching or attachment of blastocysts, but either one could inhibit outgrowth. Thus, it was concluded that human decidual beta1 integrin and FAK participate in this final step of implantation.

Animals↗

PubMed2002

Identification and cloning of a novel family of coiled-coil domain proteins that interact with O-GlcNAc transferase.

The abundant and dynamic post-translational modification of nuclear and cytosolic proteins by beta-O-linked N-acetylglucosamine (O-GlcNAc) is catalyzed by O-GlcNAc transferase (OGT). Here we used the yeast two-hybrid approach to identify and isolate GABA(A) receptor-associated protein, GRIF-1 (Beck, M., Brickley, K., Wilkinson, H. L., Sharma, S., Smith, M., Chazot, P. L., Pollard, S., and Stephenson, F. A. (2002) J. Biol. Chem. 277, 30079-30090), and its novel homolog, OIP106 (KIAA1042), as novel OGT-interacting proteins. The proteins are highly similar to each other but are encoded by two separate genes. Both GRIF-1 and OIP106 contain coiled-coil domains and interact with the tetratricopeptide repeats of OGT. GRIF-1 and OIP106 are modified by O-GlcNAc and therefore are substrates for OGT. However, unlike another high affinity protein substrate, such as nucleoporin p62, OIP106 and GRIF-1 co-immunoprecipitate with OGT, exhibiting stable in vitro and in vivo associations. Whereas GRIF-1 has been reported to be expressed only in excitable tissue, OIP106 is expressed in all human cell lines that were examined. Confocal and electron microscopy show that OIP106 localizes to nuclear punctae in HeLa cells and co-localizes with RNA polymerase II. Co-immunoprecipitation experiments confirm the presence of an in vivo RNA polymerase II-OIP106-OGT complex, suggesting that OIP106 may target OGT to transcriptional complexes for glycosylation of transcriptional proteins, such as RNA polymerase II, and transcription factors. Similarly, GRIF-1 may serve to target OGT to GABA(A) receptor complexes for mediating GABA signaling cascades.

Amino Acid Sequence↗

PubMed2002

Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis.

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline-rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two-fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.

Animals↗

PubMed2002

Changes in distribution of the long form of type XII collagen during chicken corneal development.

The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus.

Animals↗

PubMed2002

Distribution of sialoglycoconjugates in the rat cerebellum and its change with aging.

We examined the distribution of sialoglycoconjugates in the cerebellum of 9-week-old and 30-month-old rats using light microscopy and electron microscopy in combination with two lectins, Maackia amurensis lectin (MAL) for Sia(alpha)2-3Gal and Sambucus sieboldiana agglutinin (SSA) for Sia(alpha)2-6Gal. Each lectin showed characteristic staining patterns. In young adult rats, MAL stained a strongly granular layer, a weakly molecular layer, and the medullary lamina, while SSA more strongly stained the medullary lamina than the molecular and granular layers. After aging, different staining patterns were obtained. Intense SSA reactivity was observed in the granular layer and intense MAL reactivity was observed in the medullary lamina of the aged groups. The reactivity of Purkinje cells with MAL was downregulated in the aged rats. These results indicated that Sia(alpha)2-3Gal and Sia(alpha)2-6Gal were expressed in distinct regions of the rat cerebellum and that their expression patterns changed in the aged brain.

Aging↗

PubMed2002

Small guanosine triphospatase RhoA and Rho-associated kinase as regulators of trophoblast migration.

The small guanosine triphosphatase Rho controls cell adhesion and motility through reorganization of the actin cyto-skeleton and regulation of actomyosin contractility. Among the putative target molecules of Rho, a Rho-associated coiled coil-forming protein kinase (ROCK) is thought to participate in Rho-mediated cell adhesion and motility. In the present study, we explored the expression and function of RhoA and ROCK in human trophoblast cells. The colocalization of RhoA, cytokeratin 8/18, and cytokeratin 7 in some cells located in the decidual stromal region indicated that extravillous trophoblast cells expressed RhoA. In double staining for RhoA and ROCK in human chorionic villi, RhoA staining was strongly positive in the cytoplasm of cytotrophoblasts, whereas ROCK stained in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts. Both RhoA and ROCK were stained in cytoplasma of cultured human cytotrophoblast. Cultured human trophoblast cells contained actin stress fibers that were lost after treatment with C3, an exoenzyme produced by Clostridium botulinum. Y-27632, a selective ROCK inhibitor, suppressed RhoA-induced formation of actin stress fibers and formation of focal contact in trophoblast cells. The trophoblast reacquired actin stress fibers and focal contact after withdrawal of Y-27632. Cultured human cytotrophoblast cells from 7-9 wk of gestation migrated into a fibronectin-coated membrane. Both C3 exoenzyme and Y-27632 inhibited cytotrophoblast migration in a dose-dependent manner. In conclusion, cyto-trophoblasts express RhoA and ROCK in their cytoplasm, and RhoA-ROCK is involved in their assembly of actin stress fibers. Suppression of RhoA-ROCK reduces trophoblast migration. These findings suggest that RhoA-ROCK signaling is a key regulator of trophoblast cell migration.

ADP Ribose Transferases↗