Masahiro Oike

Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration

Cell migration is a fundamental biological process involving membrane polarization and cytoskeletal dynamics, both of which are regulated by Rho family GTPases. Among these molecules, Rac is crucial for generating the actin-rich lamellipodial protrusion, a principal part of the driving force for movement. The CDM family proteins, Caenorhabditis elegans CED-5, human DOCK180 and Drosophila melanogaster Myoblast City (MBC), are implicated to mediate membrane extension by functioning upstream of Rac. Although genetic analysis has shown that CED-5 and Myoblast City are crucial for migration of particular types of cells, physiological relevance of the CDM family proteins in mammals remains unknown. Here we show that DOCK2, a haematopoietic cell-specific CDM family protein, is indispensable for lymphocyte chemotaxis. DOCK2-deficient mice (DOCK2-/-) exhibited migration defects of T and B lymphocytes, but not of monocytes, in response to chemokines, resulting in several abnormalities including T lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. In DOCK2-/- lymphocytes, chemokine-induced Rac activation and actin polymerization were almost totally abolished. Thus, in lymphocyte migration DOCK2 functions as a central regulator that mediates cytoskeletal reorganization through Rac activation.

Volume-regulated Anion Channels Serve as an Auto/Paracrine Nucleotide Release Pathway in Aortic Endothelial Cells

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca2+ oscillations and NO production. They did not, however, affect Ca2+ oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a “permeating blocker model,” in which ATP binds with an affinity of 1.0 ± 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl− hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.

DOCK2 Is Essential for Antigen-Induced Translocation of TCR and Lipid Rafts, but Not PKC-θ and LFA-1, in T Cells

DOCK2 is a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City which are known to regulate actin cytoskeleton. DOCK2 is critical for lymphocyte migration, yet the role of DOCK2 in TCR signaling remains unclear. We show here that DOCK2 is essential for TCR-mediated Rac activation and immunological synapse formation. In DOCK2-deficient T cells, antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, to the APC interface was severely impaired, resulting in a significant reduction of antigen-specific T cell proliferation. In addition, we found that the efficacy of both positive and negative selection was reduced in DOCK2-deficient mice. These results suggest that DOCK2 regulates T cell responsiveness through remodeling of actin cytoskeleton via Rac activation.

Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells

1 Hypotonic stress induces ATP release followed by Ca2+ oscillations in bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress‐induced ATP release. 2 Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP‐mediated Ca2+ oscillations induced by hypotonic stress. 3 ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre‐treating the cells with Y‐27632, a selective inhibitor of Rho‐kinase, also suppressed the hypotonic stress‐induced ATP release and Ca2+ oscillations, indicating that Rho‐mediated activation of Rho‐kinase may be involved in the hypotonic ATP release. 4 Hypotonic stress also induced a transient rearrangement of the actin cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y‐27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress‐induced ATP release nor the Ca2+ oscillations. 5 These results indicate that tyrosine kinase and the Rho‐Rho‐kinase pathways are involved in hypotonic stress‐induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs.

Histamine-activated, non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca2+]i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from −0.2 to −2nA at −100mV. The tonic rise in [Ca2+]i and the ionic current are partly due to Ca2+ influx. This Ca2+ entry pathway is also permeable for Ba2+ and Mn2+. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca2+ transient, suggesting that the latter is at least partially due to Ca2+ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca2+]o) was 1.5mmol/l. With 10 mmol/l [Ca2+]o it was −13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca2+-free solution (n=3 cells). The amplitude of the current in Ca2+-free solution was enhanced compared to that in 10 mmol/l [Ca2+]o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca2+ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca2+-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca2+ signals in the absence of an agonist. It is concluded that the agonist-activated Ca2+-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.

Sequential activation of RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium

We have investigated the cellular mechanisms of mechanical stress‐induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca2+ transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS‐induced ATP release, and inhibition of the ATP‐mediated Ca2+ response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA). HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS‐ and LPA‐induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho‐kinase inhibitor (Y27632) inhibited all of the HTS‐ and LPA‐induced responses. These results indicate that the activation of the RhoA/Rho‐kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress‐specific.

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca2+ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl− current, releases Ca2+ from intracellular stores and activates Ca2+ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 μmol/l), or the F-actin-depolymerizing substance cytochalasin B (50 μmol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 μmol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 μmol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl− current. Ca2+ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca2+ stores by thapsigargin renders the intracellular [Ca2+] sensitive to the extracellular [Ca2+], which is indicative of a Ca2+ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca2+ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca2+ release by mechanical activation. The tubulin network is not involved. The Ca2+ release-activated Ca2+ entry is not modulated by the F-actin cytoskeleton.

Dual action of FRC8653, a novel dihydropyridine derivative, on the Ba2+ current recorded from the rabbit basilar artery.

Actions of FRC8653 on the macroscopic and unitary Ba2+ currents were studied using the rabbit basilar artery. Application of (+/-)-FRC8653 (less than 1 microM) increased the amplitude of the inward current when depolarization pulses more negative than -10 mV were applied but inhibited it when depolarization was more positive than 0 mV (in each case from a holding potential of -80 mV). At a holding potential of -40 mV, (+/-)-FRC8653 (greater than 0.1 nM) consistently inhibited the inward current. (-)-FRC8653 (greater than 1 nM) inhibited the amplitude of the inward current evoked by a depolarizing pulse more positive than -10 mV (the holding potential being -80 mV). At the holding potential of -80 mV, but not at -40 mV, (+)-FRC8653 (1 microM) enhanced the current amplitude evoked by a depolarizing pulse more negative than -10 mV but inhibited the current evoked by a pulse more positive than 0 mV. (+/-)-FRC8653 shifted the voltage-dependent inhibition curves to the left, and the slope of the curve became steeper (test pulse of +10 mV). Two types of single Ca2+ channel currents (12 and 23 pS) were recorded from the basilar artery by the cell-attached patch-clamp method. Opening of the 12-pS channel occurred with a depolarizing pulse (-20 mV) from a holding potential of -80 mV, but not from one of -60 mV. (+)-FRC8653 activated, and (-)-FRC8653 inhibited, the 23-pS channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Ca2+ signalling by p130, a phospholipase-C-related catalytically inactive protein: Critical role of the p130 pleckstrin homology domain

p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P(3). Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca(2+) signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca(2+) concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P(3); the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)