Sphingosine Kinase 1 Regulates the Akt/FOXO3a/Bim Pathway and Contributes to Apoptosis Resistance in Glioma Cells

The aim of this study was to investigate the mechanism through which Sphingosine kinase-1 (SPHK1) exerts its anti-apoptosis activity in glioma cancer cells. We here report that dysregulation of SPHK1 alters the sensitivity of glioma to apoptosis both in vitro and in vivo. Further mechanistic study examined the expression of Bcl-2 family members, including Bcl-2, Mcl-1, Bax and Bim, in SPHK1-overexpressing glioma cells and revealed that only pro-apoptotic Bim was downregulated by SPHK1. Moreover, the transcriptional level of Bim was also altered by SPHK1 in glioma cells. We next confirmed the correlation between SPHK1 and Bim expression in primary glioma specimens. Importantly, increasing SPHK1 expression in glioma cells markedly elevated Akt activity and phosphorylated inactivation of FOXO3a, which led to downregulation of Bim. A pharmacological approach showed that these effects of SPHK1 were dependent on phosphatidylinositol 3-kinase (PI3K). Furthermore, effects of SPHK1 on Akt/FOXO3a/Bim pathway could be reversed by SPHK1 specific RNA interference or SPHK1 inhibitor. Collectively, our results indicate that regulation of the Akt/FOXO3a/Bim pathway may be a novel mechanism by which SPHK1 protects glioma cells from apoptosis, thereby involved in glioma tumorigenesis

Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the “hydrophobic motif” of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells

In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody

Background: S1P 3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtypeselective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist. Methodology/Principal Findings: Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts. Conclusions/Significance: We have developed the first-reported monoclonal antibody that selectively recognizes a lipidactivated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsi

Phospholipase Cε is a nexus for Rho and Rap-mediated G protein-coupled receptor-induced astrocyte proliferation

Phospholipase Cε (PLCε) has been suggested to transduce signals from small GTPases, but its biological function has not yet been clarified. Using astrocytes from PLCε-deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, sphingosine 1-phosphate, and thrombin regulate phosphoinositide hydrolysis primarily through PLCε. Stimulation by lysophospholipids occurs through Gi, whereas thrombin activates PLC through Rho. Further studies reveal that PLCε is required for thrombin- but not LPA-induced sustained ERK activation and DNA synthesis, providing a novel mechanism for GPCR and Rho signaling to cell proliferation. The requirement for PLCε in this pathway can be explained by its role as a guanine nucleotide exchange factor for Rap1. Thus, PLCε serves to transduce mitogenic signals through a mechanism distinct from its role in generation of PLC-derived second messengers

(S)-FTY720-vinylphosphonate, an analogue of the immunosuppressive agent FTY720, is a pan-antagonist of sphingosine 1-phosphate GPCR signaling and inhibits autotaxin activity

FTY720 (Fingolimod), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P(1) receptor internalization and targeting to the proteasomal degradative pathway, and thus functions as an antagonist of S1P(1) by depleting the functional S1P(1) receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)- and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P(1) (EC(50) 20+/-3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P(1,3,4) (K(i) 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P(2), and S1P(5). Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K(i) values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)- and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P(2) transcripts and traces of S1P(5). (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. The biological effects of the (R)- and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets