Differential fMet-Leu-Phe- and Platelet-activating Factor-induced Signaling Toward Ral Activation in Primary Human Neutrophils

109

Store-operated calcium entry (SOCE) is a fundamental mechanism of calcium signaling. The mechanisms linking store depletion to SOCE remain controversial, hypothetically involving both diffusible messengers and conformational coupling of stores to channels. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that can signal via cell surface G-protein-coupled receptors, but S1P can also act as a second messenger, mobilizing calcium directly via unknown mechanisms. We show here that S1P opens calcium entry channels in human neutrophils (PMNs) and HL60 cells without prior store depletion, independent of G-proteins and of phospholipase C. S1P-mediated entry has the typical divalent cation permeability profile and inhibitor profile of SOCE in PMNs, is fully inhibited by 1 M Gd 3؉ , and is independent of [Ca 2؉ ] i . Depletion of PMN calcium stores by thapsigargin induces S1P synthesis. Inhibition of S1P synthesis by dimethylsphingosine blocks thapsigargin-, ionomycin-, and platelet-activating factor-mediated SOCE despite normal store depletion. We propose that S1P is a "calcium influx factor," linking calcium store depletion to downstream SOCE.Store-operated calcium entry (SOCE) 1 is the primary mechanism of regulated calcium entry into "non-excitable" cells like immunocytes. The mechanisms governing SOCE are controversial but have centered on two main hypotheses (1). In one model, emptying of endoplasmic reticulum (ER) calcium stores leads to the release of small diffusible messenger molecules that act on channels to increase Ca 2ϩ entry (2). Randriamampita and Tsien (3) found strong suggestions of such a messenger molecule in extracts from Jurkat T-cells. They termed this molecule "calcium influx factor" (CIF).Other hypotheses suggest that store depletion leads to calcium channel activation via direct physical interactions between cell membranes and intracellular structures. The "exocytosis model" (4, 5) of physical interactions suggests that vesicles bearing calcium channels fuse with the cell membrane after store depletion. The "conformational coupling" model suggests that ER calcium store depletion can lead to direct physical interactions between ER proteins such as the IP 3 receptor and cell membrane calcium channels such as TRPC3 (6, 7). No specific molecular mechanisms have been proposed however, that might initiate such physical interactions. Moreover, data has continued to accumulate supporting the existence of an unidentified, low molecular weight CIF (8, 9).Sphingosine 1-phosphate (S1P) is a universally bioactive, small (M r 380) sphingolipid metabolite. S1P is derived from the metabolism of sphingolipids and, most notably, from the actions of sphingosine kinase (10, 11) on sphingosine. In 1991, Zhang et al. (12) showed that S1P induces proliferation in Swiss 3T3 fibroblasts. Since that time, the actions of S1P have been studied in detail, and it has been found to be an important extracellular messenger molecule affecting cell growth, differentiation, adhesion, motility, and survival in many organ...

Section: S1p Induction Of Calcium Influx Is Independent Of Er Storementioning

confidence: 96%

Sphingosine 1-Phosphate, a Diffusible Calcium Influx Factor Mediating Store-operated Calcium Entry

Itagaki

Hauser

2003

109

“…Most of the inhibitors used had been shown to affect activation of Rap1. In certain cell types, an increase in [Ca 2ϩ ] i was demonstrated to be necessary or even sufficient to induce Rap1 activation and could be blocked by BAPTA-AM (1,4). Pretreatment of either MEG-01 or DAMI cells with a combination of BAPTA-AM and EGTA did not inhibit turbulence-induced Rap1GTP increase (Fig.…”

Section: Rap1 Is Activated By Turbulence and Regulates Integrinmentioning

confidence: 99%

“…2a). PKC inhibitors (Roche 31-8220, GF 109203X, staurosporine) were described as blocking block Rap1 activation in various cell types (4,6,8); however, they also did not inhibit mixing-induced elevation of active Rap1 (Fig. 2b) in these megakaryocytic cell lines.…”

Section: Rap1 Is Activated By Turbulence and Regulates Integrinmentioning

confidence: 99%

See 1 more Smart Citation

The Small GTPase Rap1 Is Activated by Turbulence and Is Involved in Integrin αIIbβ3-mediated Cell Adhesion in Human Megakaryocytes

Bruyn

Zwartkruis

Rooij

et al. 2003

Self Cite

The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrinmediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin ␣ IIb ␤ 3 , in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by ␣-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls ␣ IIb ␤ 3 -mediated cell adhesion.The small GTPase Rap1 is a molecular switch that cycles between an inactive GDP and active GTP-bound conformation. The protein can be activated by a plethora of stimuli (1-6), indicating that Rap1 activation is a common event in signaling. In general this activation is mediated by second messengers such as cAMP, calcium ions, and diacylglycerol; and a number of guanine nucleotide exchange factors have been identified to mediate this activation (7). In human platelets, Rap1 is abundantly expressed and is rapidly activated by a large variety of agonists including ␣-thrombin, thromboxane A 2 , epinephrine, platelet-activating factor, ADP, and the phorbol ester TPA 1 (1). At least two different signaling pathways are involved in this activation, one mediated by G q , phospholipase C, and calcium and one mediated by protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) (1, 8 -10). In platelets, Rap1 activation is rapidly inhibited by agents that increase the intracellular cAMP concentration, like for instance prostaglandin I 2 (1). Activation of Rap1 is prior to platelet activation, suggesting a key role of Rap1 in this process. After platelet activation Rap1 relocalizes to the actin cytoskeleton (8,11,12). In the control of Rap1 in platelets, RapGAP, which can interact with the active ␣ subunit of the heterotrimeric G-protein G z , plays a role as well (13).In a variety of cell lines, Rap1 was found to be involved in the regulation of integrin-mediated cell adhesion. Rap1 controls T-cell receptor-, CD31-, and CD98-induced activation of ␣ L ␤ 2 (14 ...

“…Rap1, another Ras-related GTPase, is rapidly and transiently activated upon stimulation by fMLP, platelet-activating factor, granulocyte-macrophage colony-stimulating factor, and IgG-coated particles. Rap1 activation is, however, independent of the respiratory burst (20).…”

mentioning

confidence: 99%

Rho Is Involved in Superoxide Formation during Phagocytosis of Opsonized Zymosans

Kim

Diebold

Kim

et al. 2004

Phagocytosis is accompanied by the production of superoxide by the NADPH oxidase complex, for which GTP-bound Rac is essential. We wanted to determine whether Rho is also involved in the production of superoxide during phagocytosis. Inhibition of Rho by Tat-C3 exoenzyme (Tat-C3) blocked superoxide formation and curtailed the phagocytosis of serum-(SOZ), C3bi-(COZ), and IgG-opsonized zymosan (IOZ) particles. Tat-C3 did not affect superoxide formation in response to phorbol myristate acetate (PMA), formyl Met-Leu-Phe (fMLP), or macrophage colony-stimulating factor (M-CSF). Superoxide formation was also reduced in J774 cells transfected with a cDNA expressing dominant-negative form of RhoA (N19RhoA). However, purified prenylated recombinant RhoA did not activate NADPH oxidase in vitro, suggesting that Rho does not interact directly with NADPH oxidase. Tat-C3 inhibited the activity of RhoA, but did not affect that of Rac in vitro or in vivo. It also inhibited the phosphorylation of p47 PHOX , one of the cytosolic components of NADPH oxidase. Taken together, these results suggest that Rho plays an important role in superoxide formation during phagocytosis of SOZ, COZ, and IOZ via phosphorylation of p47 PHOX