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K Kaibuchi

Publications and source records attributed to K Kaibuchi.

At least 19 recordsLinked to original sources
PubMed2005

Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival.

Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.

Amino Acid Sequence↗
PubMed2005

Sexually dimorphic expression of Usp9x is related to sex chromosome complement in adult mouse brain.

We found the expression of Usp9x, an X-linked gene which encodes a ubiquitin protease implicated in synaptic development, to be significantly higher in the adult female mouse brains than in male brains. The sex difference in expression of Usp9x was localized to specific brain regions such as neocortex. Furthermore, in gonadally intact and gonadectomized mice, XX mice expressed Usp9x mRNA and protein more highly than XY mice irrespective of their gonadal type. No sex difference was found in the neonatal brain or peripheral tissues such as the adult kidney. This finding implies that the difference in sex chromosome complement between XY males and XX females could potentially contribute to sexual differentiation of brain structure and function. The relation of genomic dose and Usp9x expression could help explain the neural and behavioural phenotype of women with XO Turner syndrome.

Animals↗
PubMed2001

Identification of sonic hedgehog-responsive genes using cDNA microarray.

Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-alpha-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

Animals↗
PubMed2001

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

Amino Acid Sequence↗
PubMed2001

Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts.

To clarify the mechanism of the stimulatory effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on bone formation, we investigated the effect of pitavastatin, a newly developed statin, on expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin in primary cultured human osteoblasts. Pitavastatin increased the expression level of mRNA for BMP-2, and much more effectively for osteocalcin. This stimulatory effect was abolished by the addition of geranylgeranyl pyrophosphate, an essential molecule for prenylation of small GTP-binding proteins such as Rho GTPase, but not by inhibitors of nitric oxide synthase and various protein kinases. Pitavastatin suppressed the Rho-associated kinase (Rho-kinase) activity. Hydroxyfasudil, a specific inhibitor of Rho-kinase, increased BMP-2 and osteocalcin expression. These mRNA levels were strongly suppressed by dexamethasone, but restored by co-treatment with hydroxyfasudil. These observations suggest that the Rho-kinase negatively regulates bone formation and the inhibition of Rho and Rho-kinase pathway is the major mechanism of the statin effect on bone. Moreover, a Rho-kinase inhibitor may be a new therapeutic reagent for the treatment of osteoporosis such as glucocorticoid-induced osteoporosis.

Bone Morphogenetic Protein 2↗
PubMed2001

Role of the cytoskeleton in mediating cAMP-dependent protein kinase inhibition of the epithelial Na+/H+ exchanger NHE3.

The Na(+)/H(+) exchanger NHE3 isoform mediates the entry of Na(+) into epithelial cells of the kidney and gastrointestinal tract. Hormones and pharmacological agents that activate cAMP-dependent protein kinase A (PKA) are potent inhibitors of native and ectopically expressed NHE3 in epithelial and Chinese hamster ovary AP-1 cells, respectively. Previous studies have shown that acute inhibition is coupled to direct phosphorylation of the exchanger, but this only partly accounts for the observed effects. In this report, we show that inhibition of NHE3 activity by forskolin, an activator of adenylate cyclase, occurs without changes in surface expression of the exchanger but is associated with altered cytoskeletal structure. This effect resembles that obtained with cytochalasin D or latrunculin B, actin disrupting agents that also inhibit NHE3. Such similarities prompted us to further investigate the relationship between PKA-induced inhibition of the exchanger and changes in the actin cytoskeleton. Inhibition of NHE3 by cytochalasin D does not require PKA, because the inhibitory effect is preserved in a mutant NHE3 that is not phosphorylated by PKA and in cells pretreated with the PKA inhibitor H89. In contrast, involvement of actin in the effect of cAMP on the exchanger is supported by the following observations: (i) jasplakinolide, an F-actin stabilizer, prevents the inhibition caused by forskolin, and (ii) constitutively active forms of RhoA and Rho kinase interfere with actin disruption by forskolin and also decrease inhibition of the transporter. These results suggest that reorganization of the cytoskeleton by PKA is involved in mediating inhibition of NHE3.

Actins↗
PubMed2001

Rho-kinase--mediated contraction of isolated stress fibers.

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca(2+). However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca(2+). More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca(2+)-dependent MLCK and the Rho-kinase systems. We propose that Ca(2+) is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.

Calcium↗
PubMed2001

Cytoskeletal organization in tropomyosin-mediated reversion of ras-transformation: Evidence for Rho kinase pathway.

Tropomyosin (TM) family of cytoskeletal proteins is implicated in stabilizing actin microfilaments. Many TM isoforms, including tropomyosin-1 (TM1), are down-regulated in transformed cells. Previously we demonstrated that TM1 is a suppressor of the malignant transformation, and that TM1 reorganizes microfilaments in the transformed cells. To investigate how TM1 induces microfilament organization in transformed cells, we utilized ras-transformed NIH3T3 (DT) cells, and those transduced to express TM1, and/or TM2. Enhanced expression of TM1 alone, but not TM2, results in re-emergence of microfilaments; TM1, together with TM2 remarkably improves microfilament architecture. TM1 induced cytoskeletal reorganization involves an enhanced expression of caldesmon, but not vinculin, alpha-actinin, or gelsolin. In addition, TM1-induced cytoskeletal reorganization and the revertant phenotype appears to involve re-activation of RhoA controlled pathways in DT cells. RhoA expression, which is suppressed in DT cells, is significantly increased in TM1-expressing cells, without detectable changes in the expression of Rac or Cdc42. Furthermore, expression of a dominant negative Rho kinase, or treatment with Y-27632 disassembled microfilaments in normal NIH3T3 and in TM1 expressing cells. These data suggest that reactivation of Rho kinase directed pathways are critical for TM1-mediated microfilament assemblies.

3T3 Cells↗
PubMed2001

Role of Rho-associated kinase in neointima formation after vascular injury.

BACKGROUND: The Rho/Rho-associated kinase (Rho-kinase) system is implicated in various cellular functions, including migration, proliferation, and apoptosis. Because a possible role of the system is suggested in neointima formation after vascular injury, we sought to examine whether a new specific Rho-kinase inhibitor, Y27632, prevents neointima formation of the balloon-injured rat carotid artery, and if so, to investigate the effects of Y27632 on migration, proliferation, and apoptosis of smooth muscle cells (SMCs) in the injured artery. METHODS AND RESULTS: Y27632 was administered intraperitoneally from 1 day before to 14 days after vascular injury. Treatment with Y27632 inhibited phenylephrine-induced Rho-kinase activation in the carotid artery on the basis of immunoblotting against the phosphorylated myosin-binding subunit of myosin phosphatase. Y27632 markedly prevented neointima formation at days 7 and 14. In controls, BrdU(+) proliferating and TUNEL(+) apoptotic SMCs were transiently and coincidentally increased in the neointima, with a peak at day 7. Y27632 significantly increased the neointimal TUNEL(+) SMCs at days 7 and 14, but not BrdU(+) SMCs. Y27642 significantly decreased the number of intimal SMCs at day 4, while not affecting the number of BrdU(+) or TUNEL(+) SMCs. Reendothelialization after balloon injury was not significantly affected by Y27632 at days 7 and 14. CONCLUSIONS: Y27632 inhibited neointima formation by enhancing SMC apoptosis and probably by suppressing early SMC migration. Therefore, a role of Rho-kinase is suggested in neointima formation after vascular injury.

Amides↗
PubMed2001

Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase.

Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 microM, increases force in alpha-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in alpha-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.

Amides↗
PubMed2001

IQGAP1, a negative regulator of cell-cell adhesion, is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A.

Our previous comparative genomic hybridization (CGH) study revealed a novel amplified region at 15q26 in two cell lines established from diffuse types of gastric cancer (GC). In this amplified region, FES and IGF1R, known targets on 15q26, were located telomeric to the amplicon in the two cell lines, HSC39 and 40A, suggesting that another tumor-associated gene exists in this region. While screening expressed sequence tags (ESTs) for novel genes in this region, we identified the IQGAP1 amplification. IQGAP1 has been reported to encode a ras GAP-related protein, and its interaction with cadherin and/or beta-catenin induces a dissociation of beta-catenin from the cadherin-catenin complex, one of the mechanisms for cell-cell adhesion. Northern and Western blot analyses revealed that amplification of this gene was accompanied by corresponding increases in mRNA and protein expression. Moreover, immunocytochemical staining showed that overexpressed IQGAP1 accumulated at the membrane, suggesting its colocalization with beta-catenin. Taken together, these findings suggest that IQGAP1 may be one of the target genes in the 15q26 amplicon correlated with a malignant phenotype of gastric cancer cells, such as diffuse and invasive characteristics, through the disruption of E-cadherin-mediated cell-cell adhesion.

Carrier Proteins↗
PubMed2001

Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo.

OBJECTIVE: We recently demonstrated that Rho-kinase/ROK/ROCK is functionally upregulated at the arteriosclerotic coronary lesions and plays a key role for coronary vasospastic responses in our porcine model with interleukin (IL)-1beta. In the present study, we tested our hypothesis that Rho-kinase is involved in the pathogenesis of coronary arteriosclerosis per se in our porcine model. METHODS: Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1beta. Two weeks after the procedure, coronary stenotic lesions with constrictive remodeling and vasospastic response to serotonin were noted at the IL-1beta-treated site, as previously reported. Then, animals were randomly divided into two groups; one group was treated with fasudil for 8 weeks followed by 1 or 4 weeks of washout period and another group served as a control. After oral absorption, fasudil is metabolized to hydroxyfasudil that is a specific inhibitor of Rho-kinase. RESULTS: In the fasudil group, coronary stenosis and vasospastic response were progressively reduced in vivo, while the coronary hyperreactivity was abolished both in vivo and in vitro. Furthermore, Western blot analysis showed that in the fasudil group, the Rho-kinase activity (as evaluated by the extent of phosphorylation of myosin binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase) was significantly reduced, while histological examination demonstrated a marked regression of the coronary constrictive remodeling. CONCLUSIONS: These results indicate that Rho-kinase is substantially involved in constrictive remodeling and vasospastic activity of the arteriosclerotic coronary artery, both of which could be reversed by long-term inhibition of the molecule in vivo. Thus, Rho-kinase may be regarded as a novel therapeutic target for arteriosclerotic vascular disease.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2001

Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells.

Hypercontraction or abnormal contraction of vascular smooth muscle is a major cause of diseases such as hypertension and vasospasm of the coronary and cerebral arteries. A better understanding of the mechanism of regulation of smooth muscle contraction should lead to improved treatments for such diseases. Recent studies have revealed important roles for the small GTPase Rho and its effector, Rho-associated kinase (Rho kinase) in Ca2+ independent regulation of smooth muscle contraction. The Rho-Rho-kinase pathway modulates the level of phosphorylation of the myosin light chain of myosin II, mainly through inhibition of myosin phosphatase, and contributes to agonist-induced Ca2+ sensitization in smooth muscle contraction. Rho-Rho-kinase mechanisms also participate in a variety of the cellular functions of non-muscle cells, such as stress-fibre formation, cytokinesis and cell migration. This review summarizes the role of the Rho-Rho-kinase pathway in contractile processes of smooth muscle and in non-muscle cell functions, and the pathophysiological implications of this pathway.

Animals↗
PubMed2001

FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development.

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.

Animals↗
PubMed2001

Exclusion of CD43 from the immunological synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin.

Formation of the immunological synapse requires TCR signal-dependent protein redistribution. However, the specific molecular mechanisms controlling protein relocation are not well defined. Moesin is a widely expressed phospho-protein that links many transmembrane molecules to the cortical actin cytoskeleton. Here, we demonstrate that TCR-induced exclusion of the large sialoprotein CD43 from the synapse is an active event mediated by its reversible binding to moesin. Our results also reveal that relocalization of moesin is associated with changes in the phosphorylation status of this cytoskeletal adaptor protein. Finally, these findings raise the possibility that the change in moesin localization resulting from TCR engagement modifies the overall topology of the lymphocyte membrane and facilitates molecular interactions at the site of presenting cell contact.

Animals↗
PubMed2001

Rho-family GTPases in cadherin-mediated cell-cell adhesion.

Cell-cell adhesions are rearranged dynamically during tissue development and tumour metastasis. Recently, Rho-family GTPases, including RhoA, Rac1 and Cdc42, have emerged as key regulators of cadherin-mediated cell-cell adhesion. Following the identification and characterization of regulators and effectors of Rho GTPases, signal transduction pathways from cadherin to Rho GTPases and, in turn, from Rho GTPases to cadherin, are beginning to be clarified.

Animals↗
PubMed2001

CRMP-2 induces axons in cultured hippocampal neurons.

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.

Animals↗