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H Matsushime

Publications and source records attributed to H Matsushime.

At least 19 recordsLinked to original sources
PubMed2003

Response to ADP-ribose by activation of TRPM2 in the CRI-G1 insulinoma cell line.

The response to intracellular ADP-ribose in the rat CRI-G1 insulinoma cell line was studied using a patch-clamp method. Dialysis of ADP-ribose into cells induced a response in a dose-dependent manner. The reversal potentials in various solutions showed that the ADP-ribose-gated channel was a Ca2+-permeable nonselective cation channel. In inside-out recordings, ADP-ribose and b-NAD induced responses in the same patch. The single-channel current-voltage relationships for ADP-ribose- and b-NAD-induced responses were almost identical, indicating that ADP-ribose and b-NAD activated the same channel. The physiological properties of the ADP-ribose-gated channel are similar to those we reported previously for the cloned transient receptor potential channel TRPM2. Moreover, RT-PCR analysis showed that TRPM2 was abundantly expressed in CRI-G1 cells, suggesting that the ADP-ribose-gated channel represents the native TRPM2 channel in CRI-G1 cells. These results suggest that ADP-ribose can be an endogenous modulator of Ca2+ influx through the TRPM2 channel into CRI-G1 cells.

Adenosine Diphosphate Ribose↗
PubMed2001

Functional characterization of cysteinyl leukotriene CysLT(2) receptor on human coronary artery smooth muscle cells.

Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.

Arteries↗
PubMed2001

Immunocyte Ca2+ influx system mediated by LTRPC2.

We characterized an activation mechanism of the human LTRPC2 protein, a member of the transient receptor potential family of ion channels, and demonstrated that LTRPC2 mediates Ca2+ influx into immunocytes. Intracellular pyrimidine nucleotides, adenosine 5'-diphosphoribose (ADPR), and nicotinamide adenine dinucleotide (NAD), directly activated LTRPC2, which functioned as a Ca2+-permeable nonselective cation channel and enabled Ca2+ influx into cells. This activation was suppressed by intracellular adenosine triphosphate. These results reveal that ADPR and NAD act as intracellular messengers and may have an important role in Ca2+ influx by activating LTRPC2 in immunocytes.

ADP-ribosyl Cyclase↗
PubMed2001

Rapid downregulation of cyclin D1 mRNA and protein levels by ultraviolet irradiation in murine macrophage cells.

DNA damage causes G1 cell cycle arrest through stabilization of p53 and its induction. As this process requires transcription, it takes several hours to achieve cell cycle arrest. We observed that ultraviolet (UV) light induces an immediate G1 arrest by rapid clearance of cyclin D1 in the murine macrophage cell line Bac1.2F5. The rapid disappearance of the cyclin D1 protein after exposure to UV was caused by at least two different mechanisms. In the first mechanism, cyclin D1 mRNA promptly disappeared within 1 min after UV irradiation, although cdk4 mRNA levels were unchanged. In the second mechanism, UV irradiation accelerated the degradation of cyclin D1 protein through the proteasome pathway. The half-life of the cyclin D1 protein was measured by pulse chase analysis and was shortened by UV light. These findings suggest that in the UV-irradiated Bac1.2F5 cells the amount of cyclin D1 protein is regulated at both the mRNA and protein levels. These two clearance mechanisms were also observed in murine bone-marrow-derived macrophages from wild type and p53 -/- mice, indicating that cyclin D1 mRNA and protein levels are independent of p53 function. This machinery might contribute to G1 cell cycle arrest and prevent cells from accumulating further DNA damage.

3' Untranslated Regions↗
PubMed2001

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor.

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.

Adenosine Triphosphate↗
PubMed2000

Cdk4 activation is dependent on the subunit rearrangement in the complexes.

Although several factors have been implicated in the regulation of Cdk4 activity, little is known regarding the contributions of cyclin-dependent kinase inhibitors (CKIs) in Cdk4 activation in the mid G1 phase. Using a mouse macrophage cell line (Bac1.2F5), we found that most of Cdk4 bound to p15 when cells were in a quiescent state. Following CSF-1 stimulation, Cdk4 bound to cyclin D1 and then to p21, concomitant with the dissociation of p15 from the complexes. The activation of Cdk4 correlated well with p21 binding to the complexes, and the majority of active Cdk4 complexes contained p21. During regeneration of mouse liver after partial hepatectomy, Cdk4 activity coincided precisely with ternary complex formation of cyclin D1/Cdk4/p21. Using the baculovirus expression system, we succeeded in reconstituting a capacity for Cdk4 activation in insect cells, forming an active cyclin D1/Cdk4/p21 ternary complex. Taken together, it is suggested that p21 and cyclin D1 act cooperatively as activators of Cdk4 through the release of CKIs of the INK4 family.

Animals↗
PubMed1999

Direct interaction of p21 cyclin-dependent kinase inhibitor with the retinoblastoma tumor suppressor protein.

The p21CKI forms a physical complex with the retinoblastoma protein (pRb) both in vitro and in vivo. The A/B pocket region of pRb and the N-terminal region of p21 were indispensable for this interaction. Among p21 family members, p57, but not p27, associated with pRb. Overexpression of cyclin D1, Cdk4, and E2F1 in the cells expressing pRb and p21 did not perturb the interaction between p21 and pRb. Coexpression of p21 in cells expressing pRb, cyclin D1, and Cdk4 prevented pRb hyperphosphorylation by cyclin D1/Cdk4. On the other hand, hyperphosphorylation of pRb by an excess amount of cyclin/Cdk disrupted pRb/p21 complex formation in vitro. These findings suggest that pRb may be dynamically regulated by the relative binding and activities of p21 and Cdks.

Animals↗
PubMed1998

Genomic organization of the flt-1 gene encoding for vascular endothelial growth factor (VEGF) receptor-1 suggests an intimate evolutionary relationship between the 7-Ig and the 5-Ig tyrosine kinase receptors.

The flt-1 tyrosine kinase gene encodes a high affinity receptor for Vascular Endothelial Growth Factor, and belongs to the so-called '7-Ig' or flt gene family which has characteristics of 7-Immunoglobulin (Ig)-like domains in the extracellular region. This is structurally distantly related to 5-Ig domain-containing receptors such as Fms/Kit/PDGF-R. However, the whole genomic organization for any 7-Ig receptor gene has not been determined yet. To examine the genomic structure of flt-1 and the evolutionary relationship between genes of the 7-Ig and 5-Ig receptor families, we isolated the mouse genomic DNAs carrying all exons of the flt-1 gene. The mouse flt-1 gene consisted of 30 exons, whose exon-intron boundaries were highly related to those in the 5-Ig receptor genes, except for the amino terminal region. The sequences corresponding to the first and second Ig-domains in the flt-1 gene were encoded by four exons, whereas this region was encoded by only two exons in the 5-Ig receptor genes. These results raise the interesting possibility that deletion or insertion mutations of introns in one of these receptor genes took place in the evolutionary generation of the other receptor genes.

Amino Acid Sequence↗
PubMed1997

Inactivation of the cyclin D-dependent kinase in the rat fibroblast cell line, 3Y1, induced by contact inhibition.

Cyclin-dependent kinase (Cdk) inhibitory proteins are involved in cell cycle arrest induced by antiproliferating factors or chemicals. High cell density also induces cell cycle arrest in which the genomic DNA is unreplicated, even in the presence of a mitotic dose of growth factors; this is termed contact inhibition. Although the cell cycle of the rat fibroblast cell line, 3Y1, was arrested in quiescence by contact inhibition, the Cdk4 bound to its regulatory subunit, cyclin D1 or D3. However, these complexes were enzymatically inactive. Phosphorylation of the cyclin D1-bound Cdk4 by the Cdk-activating kinase could convert the inactive cyclin D1-Cdk4 complex into its active form in vitro, suggesting that threonine 172 of the Cdk4, of which phosphorylation is required for its activation, was in part unphosphorylated in contact-inhibited 3Y1 cells. Although MO15 was active in cell extracts prepared from the arrested 3Y1 cells, activation of bacterially produced Cdk4 in the cell extracts was inhibited. Removal of p27(kip1) from the cell extracts allowed the MO15 holoenzyme to phosphorylate the Cdk4 and in turn activate it, indicating that p27(kip1) plays a role in inhibiting the phosphorylation of Cdk4 by MO15 in the contact-inhibited 3Y1 cells.

Animals↗
PubMed1997

Two different bindings of p21 Cdk inhibitor to cyclin/Cdk complex.

The cyclin-dependent kinase inhibitor p21(p21 CKI) has been found to inhibit the activity of several Cdks. Of interest wre reports that more than one molecule of p21 CKI appear to be necessary for Cdk kinase inhibition. In this report, we first determined the two different regions of p21CKI that were important for binding to cyclin D1/Cdk4 complex. Mutant analysis revealed that the either binding site is enough for their binding but for Cdk4 kinase inhibition both sites are necessary. Since the mutants (delta 17-22 or W49G) which lacks either binding site could complement each other for kinase inhibition, two molecules of p21 CKI with different binding mode might be a mechanism of cyclin D1/Cdk4 kinase inhibition.

Animals↗
PubMed1997

Contact inhibition-induced inactivation of the cyclin D-dependent kinase in rat fibroblast cell line, 3Y1.

Inhibitory proteins for Cdks (CKIs) are involved in cell cycle arrest induced by anti-mitotic factors, chemicals, or DNA damage in mammalian cells. High cell density also induces cell cycle arrest with unreplicated genomic DNA even in the presence of mitotic dose of the growth factors, termed contact inhibition. Although rat fibroblast cell line, 3Y1, arrested in quiescence by contact inhibition, Cdk4 bound its regulatory subunit, cyclin D1 or D3. However, these complexes were enzymatically inactive. Phosphorylation of the cyclin D1-bound Cdk4 by Cdk4-activating kinase, composed of cyclin H and MO15 (alias Cdk7), which was reconstituted in Spodoptera frugiperda cells (Sf9) could convert inactive cyclin D1-Cdk4 complex into active form in vitro, suggesting that threonine 172 in the Cdk4, whose phosphorylation is required for its activation, was in part unphosphorylated in the contact-inhibited 3Y1. Although MO15 was active in cell extracts prepared from the contact-inhibited 3Y1, activation of bacterially produced Cdk4 in the cell extracts was inhibited. Removing p27kip1 from the cell extracts allowed MO15 holoenzyme to phosphorylate the Cdk4 and to activate it, indicating that an access of MO15 to Cdk4 was inhibited by p27kip1 in the contact-inhibited 3Y1.

Animals↗
PubMed1996

HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4.

Tax, a regulatory protein of human T-cell leukemia virus type 1 (HTLV-1), is an oncoprotein which immortalizes human T cells and induces tumors in transgenic mice. These effects may be due to its interaction with cellular proteins, consisting of several transcription factors including CREB, NF-kappa B and SRF, and the transcriptional inhibitor, I kappa B. Here, we found that Tax binds to a cyclin-dependent kinase inhibitor, p16INK4A, which has ankyrin motifs similar to I kappa B. p16INK4A binds to the cyclin-dependent kinases, CDK4 and CDK6, and inhibits their activity, resulting in suppression of G1 phase progression. The binding of Tax to p16INK4a induced a reduction in the p16INK4A-CDK4 complex, with subsequent activation of CDK4 kinase. Tax also suppressed p16INK4A-mediated inhibition of U2OS cell growth. The p16INK4A gene was frequently deleted in many T-cell lines, but not in HTLV-1-infected T-cell lines. Taking these findings together, the functional inactivation of p16INK4A by Tax through protein-protein interaction is suggested to contribute to cellular immortalization and transformation induced by HTLV-1 infection.

Animals↗
PubMed1995

Induction of G1 arrest by down-regulation of cyclin D3 in T cell hybridomas.

The relationship between activation-induced growth inhibition and regulation of the cell cycle progression was investigated in T cell hybridomas by studying the function of the cell cycle-regulating genes such as G1 cyclins and their associated kinases. Activation of T cell hybridomas by anti-T cell receptor antibody induces growth arrest at G1 phase of the cell cycle and subsequently results in activation-driven cell death. Rapid reduction of both messenger RNA and protein level of the cyclin D3 is accompanied by growth arrest upon activation. Although the residual cyclin D3 protein forms a complex with cdk4 protein, cyclin D3-dependent kinase activity is severely impaired. Stable transfectants engineered to express cyclin D3 override the growth arrest upon activation. These results imply that the activation signal through T cell receptor induces the down-regulation of cyclin D3 expression and cyclin D3-dependent kinase activity, leading to growth arrest in G1 phase of the cell cycle in T cells.

Animals↗
PubMed1995

[Cell cycle and parathyroid tumor].

Temporally orderly activation of cyclin-dependent kinases (Cdks) governs progression and transitions of the cell cycle in eukaryotic cells. Binding of Cdks to cyclins and threonine phosphorylation in the Cdks are required to form fully active holo-Cdks. So far, 8 types of cyclins and 7 Cdks are known in mammals. Two types of the cyclins, D-(D1, D2, D3) and E-type cyclins, function in the G1 phase of the cell cycle. D-type cyclins form active complexes with Cdk 4 or Cdk 6 earlier than E-type cyclin does with Cdk 2. Overexpression of the cyclin D1 has been reported in human tumors including parathyroid adenomas with chromosomal abnormality, suggesting that overexpression of the cyclin D1 could abrogate cell cycle control in G1 phase and may contribute to generate tumor cells.

Animals↗
PubMed1995

[Macrophage cell cycle control by M-CSF/CSF-1].

Temporally orderly activation of the cyclin-dependent kinases (cdks) governs the progression and the transitions of the cell cycle in mammalian cells. Macrophages require a specific growth factor, M-CSF/CSF-1, for their proliferation throughout the G1 phase of the cell cycle. Once cells enter S phase, macrophages complete mitosis in the absence of M-CSF/CSF-1. During the G1 phase, cyclin D1 is induced by M-CSF/CSF-1 stimulation and forms enzymatically active complex with cdk 4. The enzymatic activity of the cyclin D1 and cdk 4 complex could be negatively regulated by recently reported inhibitory proteins to determine the timing for entry into S phase in macrophages.

Animals↗
PubMed1994

D-type cyclin-dependent kinase activity in mammalian cells.

D-type cyclin-dependent kinase activities have not so far been detected in mammalian cells. Lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3 or to their major catalytic partner, cyclin-dependent kinase 4 (cdk4), yielded kinase activities in immune complexes which readily phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. Virtually all cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts could be attributed to cdk4. When quiescent cells were stimulated by growth factors to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 phase lagged significantly behind cyclin induction and correlated with the more delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition, which occurs within the first few hours following growth factor stimulation. Rodent fibroblasts engineered to constitutively overexpress either cyclin D1 alone or cyclin D3 together with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Therefore, despite continued enforced overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and the appearance of their kinase activities remained dependent upon serum stimulation, indicating that upstream regulators must govern formation of the active enzymes.

3T3 Cells↗
PubMed1994

Possible involvement of VEGF-FLT tyrosine kinase receptor system in normal and tumor angiogenesis.

A novel receptor-type tyrosine kinase gene flt (fms-like tyrosine kinase, flt-1) was isolated from human placenta cDNA library. Flt-1 receptor carries a ligand binding domain which contains seven immunoglobulin-like stretches. Further, Flt-1 tyrosine kinase domain is separated by an approximately 70 amino acid-insert region similar to the cases of Fms/Kit/PDGF-R (fms family). However, unlike the fms family members, Flt-1 insert region does not contain "Tyr-X-X-Met" motif, which is known to be important for signal transduction and for the binding of P13 kinase to the receptor. Thus, a unique structure of Flt-1 suggests that a signal transduction pathway from Flt receptor is different from that in the fms family. The expression of flt-1 gene is detectable in a variety of normal tissues, and cell fractionation studies indicate that the flt-1 mRNA is highly expressed in endothelial cell-enriched fraction. VEGF, which has recently been reported as a ligand for Flt-1 receptor, dramatically stimulated growth of endothelial cells in culture. Many human tumors were found to express VEGF mRNA but not Flt-1 message, suggesting a paracrine mechanism for tumor angiogenesis. Interestingly, VEGF could not stimulate proliferation of Flt-1-overexpressing NIH3T3 fibroblast cells. These results suggest that Flt-1 is an endothel-specific growth factor receptor and that the signal transducing pathway through Flt-1 receptor is inactive in the fibroblast background.

3T3 Cells↗