Search PubMed⌕ Search

Biomedical subjects

K Sobue

Publications and source records attributed to K Sobue.

At least 55 records · Page 3Linked to original sources
PubMed1997

Smooth muscle cell phenotype-dependent transcriptional regulation of the alpha1 integrin gene.

The expressional regulation of chicken alpha1 integrin in smooth muscle cells was studied. The alpha1 integrin mRNA was expressed developmentally and was distributed dominantly in vascular and visceral smooth muscles in chick embryos. In a primary culture of smooth muscle cells, alpha1 integrin expression was dramatically down-regulated during serum-induced dedifferentiation. Promoter analyses revealed that the 5'-upstream region (-516 to +281) was sufficient for transcriptional activation in differentiated smooth muscle cells but not in dedifferentiated smooth muscle cells or chick embryo fibroblasts. Like other alpha integrin promoters, the promoter region of the alpha1 integrin gene lacks TATA and CCAAT boxes and contains binding sites for AP1 and AP2. The essential difference from other alpha integrin promoters is the presence of a CArG box-like motif. Deletion and site-directed mutation analyses revealed that the CArG box-like motif was an essential cis-element for transcriptional activation in differentiated smooth muscle cells, whereas the binding sites for AP1 and AP2 were not. Using specific antibodies, a nuclear protein factor specifically bound to the CArG box-like motif was identified as serum response factor. These results indicate that alpha1 integrin expression in smooth muscle cells is regulated transcriptionally in a phenotype-dependent manner and that serum response factor binding plays a crucial role in this regulation.

Animals↗
PubMed1997

Coordinate expression of alpha-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells.

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the alpha-tropomyosin (alpha-TM) gene, exon 2a rather than exon 2b is specifically spliced in alpha-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of alpha-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the down-regulation of beta-TM-SM, alpha-TM-SM converted to alpha-TM-F1 and alpha-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high Mr type to low Mr type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express alpha-TM-SM, beta-TM-SM, and high Mr caldesmon. In situ hybridization revealed specific coexpression of alpha-TM-SM and high Mr caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.

Animals↗
PubMed1997

Characterization of the interaction between synapsin I and calspectin (brain spectrin or fodrin).

We characterized the properties of the interaction between synapsin I and calspectin using purified proteins. The binding assay in the native state using antibodies specific to the tail region of synapsin I revealed that the binding is a high affinity with Kd of 9 nM, which is almost comparable to that of synapsin I to synaptic vesicles and to F-actin. We demonstrated that the head-middle region of synapsin I binds the NH2-terminal domain of beta subunit of calspectin, which also contains an actin binding site. Furthermore, the interaction was significantly inhibited by phosphorylation of synapsin I by cAMP-dependent protein kinase or by Ca2+, calmodulin-dependent protein kinase II. These properties of the interaction between synapsin I and calspectin may help understanding of its modulatory roles in neurotransmitter release.

Animals↗
PubMed1997

A defect in cell-to-cell adhesion via integrin-fibronectin interactions in a highly metastatic tumor cell line.

We investigated the role of integrin-fibronectin (FN) interactions in tumor cell adhesion. Two cloned tumor cell lines designated OV-LM (low-metastatic) and OV-HM (high-metastatic) were isolated from a murine ovarian carcinoma, OV2944. OV-LM and OV-HM cells exhibited high and low RGDS-sequence-dependent adhesiveness to FN, respectively. Both lines expressed comparable levels of alpha5 and alpha v integrins, which are capable of reacting with RGDS on FN. To compare the functions of these integrins between the two tumor lines, the signaling mechanism following FN stimulation was examined. Significant levels of phosphorylation of focal adhesion kinase (FAK) were detected in both OV-LM and OV-HM cells before FN stimulation. Whereas the level of FAK phosphorylation was appreciably enhanced in OV-LM cells stimulated with FN, stimulation of OV-HM cells with FN induced a reduction in the FAK phosphorylation in association with a significant decrease in the amount of FAK protein in the soluble compartment of cell lysates. A difference in the deposition of FN on the cell surface was also observed between the two types of tumor lines; OV-HM cells had an appreciably smaller amount of FN than OV-LM. Consistent with the functional abnormality of the integrin-FAK system and the smaller amount of FN on OV-HM, this clone exhibited a reduced cell-cell adhesion in the in vitro cell aggregation assay. Namely, OV-LM cells displayed a time-dependent increase in the formation of cell aggregates, whereas most OV-HM cells remained single. The formation of aggregates by OV-LM cells was inhibited by addition of RGDS peptide. These results indicate that the highly metastatic clone, OV-HM, exhibits a decreased capacity of cell-cell adhesion mediated by integrin-FN interactions and suggest that this defect is mainly due to the dysfunction of integrins/FAK rather than a decrease in the amount of integrins expressed on tumor cells.

Animals↗
PubMed1996

Phase specific association of heterotrimeric GTP-binding proteins with the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Subcellular distribution of heterotrimeric GTP-binding proteins during thrombin receptor-mediated platelet activation was examined, revealing two phases of translocation to the cytoskeleton. A part of Gi2 alpha and Gs alpha shows first phase translocation to the low-speed pellet (15000 x g pellet) within 1 min after activation, suggesting involvement in platelet shape change or granule secretion. In the second phase, Gi2 alpha, Gs alpha, Gq alpha, and G beta translocate to the low-speed pellet, depending on platelet aggregation. These translocations correlated with the reorganization of the actin-cytoskeleton and were inhibited by cytochalasin D. Reconstitution experiments also revealed that G proteins are associated with the actin-cytoskeleton during platelet activation.

Actins↗
PubMed1996

Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve.

The localization of synapsin I, a synaptic vesicle-associated protein, was investigated immunocyto-chemically in normal nerve fibers and regenerating axonal sprouts following crush-injuries to the rat sciatic nerve. In normal myelinated axons, weak synapsin I immunoreactivity was found in the axoplasmic/smooth endoplasmic domains, but not in the cytoskeletal domains comprising neurofilaments and microtubules. In non-myelinated axons without dense cytoskeletal structures, moderate immunoreactivity was distributed diffusely throughout the axoplasm. In the crush-injured nerves, intense synapsin I immunoreactivity was demonstrated by light microscopy in early regenerating sprouts emerging from nodes of Ranvier. These nodal sprouts subsequently elongated as regenerating axons through the space between the basal lamina and the myelin sheath (or Schwann cell plasma membrane). Intense synapsin I immunoreactivity was also found in the growth cones of such long regenerating axons. Electron microscopy revealed that synapsin I immunoreactivity was associated mainly with vesicular organelles in the nodal sprouts and growth cones of regenerating axons. Long regenerating axons exhibited no synapsin I immunoreactivity in the shaft, which contained an abundance of neurofilaments. However, vesicle accumulations remaining in the periphery of the shaft still exhibited intense synapsin I immunoreactivity. Thus, it can be concluded that synapsin I is localized at especially high density in the domains comprising vesicular organelles, which are characteristic of early nodal sprouts, as well as in growth cones of regenerating axons. These findings, together with the proposed functions of synapsin I investigated in other studies, suggest that synapsin I may play important roles in vesicular dynamics including the translocation of vesicles to the plasma membrane in sprouts and growth cones of regenerating axons.

Animals↗
PubMed1995

Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.

The intracellular protein tyrosine kinase FAK (focal adhesion kinase) was originally identified gy its high level of tyrosine phosphorylation in v-src-transformed cells. FAK is also highly phosphorylated during early development. In cultured cells it is localized to focal adhesion contacts and becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix, suggesting an important role in cell adhesion and/or migration. We have generated FAK-deficient mice by gene targeting to examine the role of FAK during development. Mutant embryos displayed a general defect of mesoderm development, and cells from these embryos had reduced mobility in vitro. Surprisingly, the number of focal adhesions was increased in FAK-deficient cells, suggesting that FAK may be involved in the turnover of focal adhesion contacts during cell migration.

Animals↗
PubMed1995

Transcriptional regulation of the chicken caldesmon gene. Activation of gizzard-type caldesmon promoter requires a CArG box-like motif.

Caldesmon, which plays a vital role in the actomyosin system, is distributed in smooth muscle and non-muscle cells, and its isoformal interconversion between a high M(r) form and low M(r) form is a favorable molecular event for studying phenotypic modulation of smooth muscle cells. Genomic analysis reveals two promoters, of which the gizzard-type promoter displays much higher activity than the brain-type promoter. Here, we have characterized transcriptional regulation of the gizzard-type promoter. Transient transfection assays in chick gizzard smooth muscle cells, chick embryo fibroblasts, mouse skeletal muscle cell line (C2C12), and HeLa cells revealed that the promoter activity was high in smooth muscle cells and fibroblasts, but was extremely low in other cells. Cell type-specific promoter activity depended on an element, CArG1, containing a unique CArG box-like motif (CCAAAAAAGG) at -315, while multiple E boxes were not directly involved in this event. Gel shift assays showed the specific interaction between the CArG1 and nuclear protein factors in smooth muscle cells and fibroblasts. These results suggest that the CArG1 is an essential cis-element for cell type-specific expression of caldesmon and that the function of CArG1 might be controlled under phenotypic modulation of smooth muscle cells.

Animals↗
PubMed1995

Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones.

Neuronal growth cones move actively, accompanying changes in intracellular Ca2+ concentration. The movement of growth cones may partly depend on the actomyosin system, considering the presence of actin and myosin II. Yet, Ca(2+)-sensitive regulatory proteins for the actomyosin system have not been identified in growth cones. In the present study, caldesmon, an inhibitory protein on actin-myosin interaction, was detected in the growth cone fraction isolated from embryonic rat brain, using immunoblotting with the antibody to chicken gizzard caldesmon. Morphological evidence of caldesmon in growth cones of cultured rat neurons was obtained using the indirect immunofluorescence method. Since inhibition of caldesmon on actin-myosin interaction can be overcome by calmodulin and Ca2+, caldesmon may be involved in the Ca(2+)-dependent regulation in growth cone motility. Tropomyosin is another member of the actomyosin system whose function may be regulated by caldesmon in smooth and nonmuscle cells. A low Mr isoform of tropomyosin was distributed in the growth cone fraction. Using specific antibodies against tropomyosin isoforms, we further clarified morphologically that the low Mr isoform was localized in growth cones, but not the high Mr isoform. High Mr isoforms of tropomyosin were present in nonneuronal cells. Actin filaments in growth cones may be unstable, since low Mr tropomyosin binds to actin filaments with a lower affinity than high Mr isoforms. The instability of actin filaments may be suitable for the rapid movement and shape changes of growth cones.

Actins↗
PubMed1995

Translocation of cortactin (p80/85) to the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Cortactin (p80/85) was discovered as a src kinase substrate and an actin filament binding protein. We investigated translocation of cortactin to the cytoskeleton during thrombin receptor-mediated platelet activation. Only a few percent of total cortactin (minor cortactin pool) translocates to the cytoskeleton as early as 5 s after platelet activation, while about 40% of total cortactin (major cortactin pool) is thereafter recovered in the cytoskeleton during platelet aggregation. Pretreatment of platelets with cytochalasin D suppresses completely this translocation, indicating that the translocation is dependent on actin polymerization. Inhibition of platelet aggregation by a tetrapeptide with the sequence RGDS, chelator of extracellular Ca2+, or a nonstirring condition results in marked suppression of translocation of the major cortactin pool. These results suggest that a minor cortactin pool translocates to the cytoskeleton independent of GPII-bIIIa (alpha IIb beta 3 integrin) engagement, and a major pool requires GPIIbIIIa-mediated signals into the cell for the translocation. Methyl 2,5-hydroxycinamate, a tyrosine kinase inhibitor, inhibits tyrosine phosphorylation of cortactin without affecting its translocation, indicating that tyrosine phosphorylation is not essential for the translocation. Morphological studies reveal that cortactin is colocalized with filamentous actin in aggregated platelets and that it is localized at the cell peripheries along actin filaments in spread platelets. Taking these together, we have demonstrated in this paper that the translocation of cortactin is associated with the reorganization of the actin-based cytoskeleton during platelet activation, particularly with platelet aggregation.

Actins↗
PubMed1995

Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin (nonerythroid spectrin or fodrin) by calpain.

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.

Animals↗
PubMed1994

Annexin VI-binding proteins in brain. Interaction of annexin VI with a membrane skeletal protein, calspectin (brain spectrin or fodrin).

Identification of annexin VI-binding proteins is essential to elucidate the physiological functions of annexin VI. Here, we developed the methods to identify an annexin VI-binding protein and characterized the binding. Annexin VI bound to about 14 species of proteins in the whole homogenate of rat forebrain, when examined with 125I-annexin VI using blots of SDS-polyacrylamide gels. The binding was Ca(2+)-dependent and specific for phosphatidylserine (PS) and phosphatidic acid. A line of evidence indicates that the binding of annexin VI to its target proteins is a protein-protein interaction. One of annexin VI-binding proteins with M(r) 240,000 was enriched in the cytoskeletal fraction and was identified as calspectin (brain spectrin or fodrin). When the binding was examined with purified calspectin in the native state, the Ca2+ affinity (KCa) was 7.6 microM, and the affinity for annexin VI (Kd) was 68 nM. Annexin VI bound to beta subunit of calspectin, but not to alpha subunit. The binding site was localized to the NH2-terminal domain of beta subunit, which contains an actin-binding site and exhibits striking homology with the NH2-terminal regions of dystrophin and alpha-actinin. When the effect of annexin VI on the interaction between F-actin and calspectin was examined by low shear viscometry, annexin VI inhibited the F-actin cross-linking activity of calspectin in a Ca2+/PS-dependent manner. Cosedimentation assay showed that annexin VI dissociates calspectin from F-actin in the presence of Ca2+ and PS. These results suggest that annexin VI can dissociate and redistribute calspectin in a Ca2+/phospholipid-dependent manner under the plasma membrane and that annexin VI may be involved in the regulation of the membrane skeleton of neuronal cells in response to Ca2+.

Actins↗
PubMed1994

Identification of two distinct promoters in the chicken caldesmon gene.

Caldesmon (CaD) is a suitable molecular marker for phenotypic modulation of smooth muscle cells. Chicken CaD gene is composed of 17 exons with a whole length of 100-150 kilobases (kb). Exons 1a-1, 1a-2, and 1a-3 encode the 5'-terminal sequence specific to mRNA for gizzard type CaD, and exon 1b encodes the sequence specific to brain type CaD mRNA. Here, we have characterized the 5'-upstream regions of chicken CaD gene. Primer extension analysis revealed that the transcriptional starting sites of gizzard and brain CaD mRNAs were 218 and 279 nucleotides upstream from each translational initiation codon, respectively. We have identified two distinct promoters (gizzard type and brain type promoters) in the CaD gene by bacterial chloramphenicol acetyltransferase (CAT) assay using chick embryo fibroblasts (CEFs). The 5'-upstream region of exon 1a-1 showed remarkable promoter activity, but the activity of the 5'-upstream region of exon 1b was low (10% of the former). These results indicate that CaD subtypes are generated by differential RNA transcriptions.

Animals↗
PubMed1994

Annexin VI binds to a synaptic vesicle protein, synapsin I.

Annexin VI bound to > 14 species of proteins in the whole homogenate of rat forebrain in a Ca2+/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M(r) 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I. Annexin VI bound to the head domain of synapsin I. When the binding of annexin VI to synapsin I was characterized in the native state, the affinity of the binding for Ca2+ (KCa) was 12.6 microM, and the affinity for annexin VI (KD) was approximately 270 nM. Phosphorylation of synapsin I by cyclic AMP-dependent protein kinase and by Ca2+/calmodulin-dependent protein kinase II inhibited the annexin VI binding. The mode of the inhibition was different between the two kinases. These results indicate that annexin VI may modulate the function of synapsin I in a Ca(2+)- and phospholipid-dependent manner.

Animals↗
PubMed1994

Localization and characterization of gelsolin in nervous tissues: gelsolin is specifically enriched in myelin-forming cells.

Gelsolin is a Ca(2+)-sensitive actin filament-severing protein. To elucidate the role of gelsolin in nervous tissues, we have investigated localization and expression of gelsolin in rat CNS and PNS using biochemical and morphological methods with a polyclonal antibody against the COOH-terminal fragment of plasma gelsolin. Immunohistochemical study showed that gelsolin was specifically enriched in oligodendrocytes and Schwann cells, and was also detected in myelin sheath, especially around the Ranvier's nodes. The immunohistochemical stainings using indirect immunofluorescence, avidin-biotin-peroxidase complex, and immunogold methods were carefully confirmed by immunoblotting against the tissue homogenates. The expressional changes of gelsolin in developing brain were investigated. The protein was detectable in newborn rat brain; however, it began to increase at 8-10 d after birth and reached maximal at 20-30 d when myelinogenesis actively occurred. After this period, the protein decreased gradually, although myelin basic protein was increasing until 6 months after birth. The immunostaining of gelsolin in Schwann cells was enhanced upon regeneration of injured sciatic nerves by freezing. Immunoelectron microscopy revealed that gelsolin was present not only in the cytoplasm but also in compact myelin. Following solubilization by detergents, gelsolin in the myelin fraction could be purified using anion exchange and blue Sepharose column chromatographies. The purified protein possessed a Ca(2+)-dependent severing activity against actin filaments similar to that of cytoplasmic and plasma gelsolin. These data strongly suggest that gelsolin in nervous tissues might be involved in lamellipodial movement to wrap axons of myelin-forming cells by modulating actin polymerization.

Animals↗
PubMed1993

Gelsolin is localized in neuronal growth cones.

Gelsolin, a Ca(2+)-sensitive actin filament-severing protein, is involved in actin turnover. Immunocytochemical study with anti-gelsolin antibody revealed that the protein is localized in both filopodia and the body part of growth cones of differentiated PC12 cells with nerve growth factor (NGF) and of rat dorsal root ganglion neurons. Here, we identified gelsolin as one of the molecular bases for the Ca(2+)-dependent movement of growth cones.

Animals↗
PubMed1993

Common structural and expressional properties of vertebrate caldesmon genes.

We have determined the genomic structure of chicken caldesmon (CaD) gene. The gene, 100-150 kilobases long, is composed of 17 exons. Exons 1a-1, 1a-2, and 1a-3 encode the 5'-terminal sequence specific to the mRNAs for CaDs expressed in gizzard. Exon 1b encodes the 5'-terminal sequence of the brain l-CaD and locates downstream of exons 1a-1, 1a-2, and 1a-3. The genomic construction of the chicken CaD resembles with that of the human CaD. Exon 3 of chicken CaD gene possesses the unique structure similar to that of human CaD gene; the common domain in both h- and l-CaDs (amino acid residues 74-199 for h-CaD and residues 66-191 for l-CaD) and the central repeating domain specific to h-CaD (amino acid residues 200-419) are encoded in exons 3a and 3b, respectively. Of particular interest is that the two consensus 5'-splice sites are found in the borders between exons 3a and 3b, and exon 3b and intron. Therefore, the expressional regulation between h- and l-CaDs can be explained by selection of these 5'-splice sites. Alternative 3'-splice sites also exist at intron/exon junction of exon 14 and the difference in selection of the sites would induce the specific Ala-508 insertion in the brain l-CaD.

Alternative Splicing↗