Shawn G. Payne

Role of ABCC1 in export of sphingosine-1-phosphate from mast cells

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses.

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis.

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.

The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2.

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G‐protein‐coupled sphingosine‐1‐phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.

Coadministration of Histone Deacetylase Inhibitors and Perifosine Synergistically Induces Apoptosis in Human Leukemia Cells through Akt and ERK1/2 Inactivation and the Generation of Ceramide and Reactive Oxygen Species

Interactions between histone deacetylase inhibitors (HDACIs) and the alkyl-lysophospholipid perifosine were examined in human leukemia cells. Coadministration of sodium butyrate, suberoylanilide hydroxamic acid (SAHA), or trichostatin with perifosine synergistically induced mitochondrial dysfunction (cytochrome c and apoptosis-inducing factor release), caspase-3 and -8 activation, apoptosis, and a marked decrease in cell growth in U937 as well as HL-60 and Jurkat leukemia cells. These events were associated with inactivation of extracellular signal-regulated kinase (ERK) 1/2 and Akt, p46 c-jun-NH2-kinase (JNK) activation, and a pronounced increase in generation of ceramide and reactive oxygen species (ROS). They were also associated with up-regulation of Bak and a marked conformational change in Bax accompanied by membrane translocation. Ectopic expression of Bcl-2 delayed but was ultimately ineffective in preventing perifosine/HDACI-mediated apoptosis. Enforced expression of constitutively active mitogen-activated protein kinase kinase (MEK) 1 or myristoylated Akt blocked HDACI/perifosine-mediated ceramide production and cell death, suggesting that MEK/ERK and Akt inactivation play a primary role in these phenomena. However, inhibition of JNK activation (e.g., by the JNK inhibitor SP600125) did not attenuate sodium butyrate/perifosine-induced apoptosis. In addition, the free radical scavenger N-acetyl-L-cysteine attenuated ROS generation and apoptosis mediated by combined treatment. Finally, the acidic sphingomyelinase inhibitor desipramine attenuated HDACI/perifosine-mediated ceramide and ROS production as well as cell death. Together, these findings indicate that coadministration of HDACIs with perifosine in human leukemia cells leads to Akt and MEK/ERK disruption, a marked increase in ceramide and ROS production, and a striking increase in mitochondrial injury and apoptosis. They also raise the possibility that combining these agents may represent a novel antileukemic strategy.

Differential transactivation of sphingosine-1-phosphate receptors modulates NGF-induced neurite extension

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

The Immunomodulator FTY720 Has a Direct Cytoprotective Effect in Oligodendrocyte Progenitors

The immunomodulator 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (FTY720) has promising therapeutic effects in multiple sclerosis (MS), a degenerative disease in which demyelination of the central nervous system is accompanied by death of oligodendrocytes (OLGs), the myelin-producing cells. In vivo phosphorylation of FTY720 generates an agonist for G protein-coupled receptors for sphingosine-1-phosphate, a lipid mediator that plays a crucial role in the stimulation of OLG survival by neurotrophin-3 (NT-3). The mechanisms underlying the action of FTY720 in MS are not clearly understood, although the effects of this drug in autoimmune diseases are thought to stem from its ability to reduce lymphocyte infiltration and inflammation. Interestingly, we now found that FTY720 also has a direct effect on OLG progenitors. Treatment of these cells with FTY720 causes activation of extracellular signal-regulated kinase 1/2 and Akt, accompanied by protection from apoptosis. However, FTY720 also arrested OLG differentiation. Importantly, this effect was counteracted by NT-3, which not only enhanced the survival of OLG progenitors induced by FTY720 but also stimulated their maturation. Altogether, these observations suggest that in addition to its immunosuppressive functions, FTY720 could also have a beneficial effect in MS by direct action on OLG progenitors. However, the finding that FTY720 blocks the differentiation of these cells raises the question of whether MS therapies with FTY720 should include the use of differentiation-enhancing factors such as NT-3. This approach would ensure both protection of existing OLG progenitor pools against immune-mediated insults as well as stimulation of remyelination by enhancing the maturation of these cells.

A novel acylglycerol kinase that produces lysophosphatidic acid modulates cross talk with EGFR in prostate cancer cells

The bioactive phospholipids, lysophosphatidic acid (LPA) and phosphatidic acid (PA), regulate pivotal processes related to the pathogenesis of cancer. Here, we report characterization of a novel lipid kinase, designated acylglycerol kinase (AGK), that phosphorylates monoacylglycerol and diacylglycerol to form LPA and PA, respectively. Confocal microscopy and subcellular fractionation suggest that AGK is localized to the mitochondria. AGK expression was up-regulated in prostate cancers compared with normal prostate tissues from the same patient. Expression of AGK in PC-3 prostate cancer cells markedly increased formation and secretion of LPA. This increase resulted in concomitant transactivation of the EGF receptor and sustained activation of extracellular signal related kinase (ERK) 1/2, culminating in enhanced cell proliferation. AGK expression also increased migratory responses. Conversely, down-regulating expression of endogenous AGK inhibited EGF- but not LPA-induced ERK1/2 activation and progression through the S phase of the cell cycle. Hence, AGK can amplify EGF signaling pathways and may play an important role in the pathophysiology of prostate cancer.

The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nm) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nm), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-β-d-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27KIP1, cyclins A, E, and D1, and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH2-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger l-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

The Histone Deacetylase Inhibitor LAQ824 Induces Human Leukemia Cell Death through a Process Involving XIAP Down- Regulation, Oxidative Injury, and the Acid Sphingomyelinase- Dependent Generation of Ceramide

Determinants of differentiation and apoptosis induction by the novel histone deacetylase inhibitor (HDACI) LAQ824 were examined in human leukemia cells (U937 and Jurkat). Exposure of U937 cells to a low concentration of LAQ824 (30 nM) resulted in a delayed (2 h) increase in reactive oxygen species (ROS), induction of p21WAF1/CIP1, pRb dephosphorylation, growth arrest of cells in G0/G1 phase, and differentiation. On the other hand, exposure of cells to a higher concentration of LAQ824 (75 nM) resulted in the early (30 min) generation of ROS, arrest of cells in G2/M phase, down-regulation of XIAP (at the transcriptional level) and Mcl-1 (through a caspase-mediated process), the acid sphingomyelinase-dependent generation of ceramide, and profound mitochondrial injury, caspase activation, and apoptosis. LAQ824-induced lethality in U937 cells did not involve the extrinsic apoptotic pathway, nor was it associated with death receptor up-regulation; instead, it was markedly inhibited by ectopic expression of Bcl-2, Bcl-xL, XIAP, and Mcl-1. The free radical scavenger N-acetyl cysteine blocked LAQ824-mediated ROS generation, mitochondrial injury, Mcl-1 down-regulation, ceramide generation, and apoptosis, suggesting a primary role for oxidative injury in LAQ824 lethality. Together, these findings indicate that LAQ824-induced lethality represents a multifactorial process in which LAQ824-mediated ROS generation is necessary but not sufficient to induce apoptosis, and that the degree of XIAP and Mcl-1 down-regulation and ceramide generation determines whether this agent engages a maturation rather than an apoptotic program.

Ceramide kinase regulates growth and survival of A549 human lung adenocarcinoma cells.

Ceramide‐1‐phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro‐apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro‐apoptotic ceramide and anti‐apoptotic C1P to regulate cell fate, reminiscent of its function in plants.