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1.  Sphingolipid metabolites in inflammatory disease 
Nature  2014;510(7503):58-67.
Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes. Progress in our understanding of sphingolipid metabolism, state-of-the-art sphingolipidomic approaches and animal models have generated a large body of evidence demonstrating that sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate, are signalling molecules that regulate a diverse range of cellular processes that are important in immunity, inflammation and inflammatory disorders. Recent insights into the molecular mechanisms of action of sphingolipid metabolites and new perspectives on their roles in regulating chronic inflammation have been reported. The knowledge gained in this emerging field will aid in the development of new therapeutic options for inflammatory disorders.
doi:10.1038/nature13475
PMCID: PMC4320971  PMID: 24899305
2.  SPHINGOSINE-1-PHOSPHATE IN CHRONIC INTESTINAL INFLAMMATION AND CANCER 
Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, and the kinase that produces it have now emerged as key regulators of numerous cellular processes involved in inflammation and cancer. Here, we review the importance of S1P in colitis and colitis-associated cancer (CAC) and discuss our recent work demonstrating that S1P produced by upregulation of SphK1 during colitis and associated cancer is essential for production of the multifunctional NF-kB-regulated cytokine IL-6, persistent activation of the transcription factor Stat3, and consequent upregulation of the S1P receptor, S1PR1. The effectiveness of the pro-drug FTY720 (known as fingolimod), approved for the treatment of multiple sclerosis, has become the gold standard for S1P-centric drugs, and will be used to illustrate the therapeutic value of modulating SphK1 and S1P receptor functions. We will discuss our recent results showing that FTY720/fingolimod administration interferes with the SphK1/S1P/S1PR1 axis and suppresses the NF-kB/IL-6/Stat3 malicious amplification loop and CAC. These preclinical studies suggest that FTY720/fingolimod may be useful in treating colon cancer in individuals with ulcerative colitis.
doi:10.1016/j.jbior.2013.10.001
PMCID: PMC3946530  PMID: 24210073
sphingosine-1-phosphate; sphingosine kinase; colitis; colitis associated cancer; inflammation; FTY720
3.  Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory 
Nature neuroscience  2014;17(7):971-980.
FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.
doi:10.1038/nn.3728
PMCID: PMC4256678  PMID: 24859201
4.  The Potential of Histone Deacetylase Inhibitors in Niemann–Pick Type C Disease 
The FEBS journal  2013;280(24):10.1111/febs.12505.
Niemann-Pick type C (NPC) disease is a fatal complex neurodegenerative lysosomal storage disorder caused by genetic mutations in either NPC1 (95% of patients) or NPC2 that decrease intracellular cholesterol trafficking resulting in accumulation of unesterified cholesterol and sphingolipids in lysosomal storage organelles. Unfortunately, treatment options for NPC disease are still very limited although miglustat, which inhibits glucosylceramide synthase, limiting ganglioside accumulation, has been approved for treatment of NPC disease. Here we discuss advances in understanding of NPC1 and its functions and several new strategies for interfering with cholesterol and sphingolipid accumulation in NPC1 null mice. We also describe several recent intriguing studies demonstrating that histone deacetylase inhibitors can correct cholesterol storage defects in human NPC1 mutant fibroblasts by increasing expression of the low transport activity NPC1 mutant protein. These studies might lead to development of new therapeutic approaches for treatment of NPC disease.
doi:10.1111/febs.12505
PMCID: PMC3857996  PMID: 23992240
Niemann–Pick type C disease; NPC1; NPC2; Cholesterol; Glycosphingolipids; HDAC
5.  Genotype-dependent Effects of TGFβ1 on Mast Cell Function: Targeting the Stat5 Pathway 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(9):10.4049/jimmunol.1202723.
We previously demonstrated that Transforming Growth Factor (TGF) β1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high affinity IgE receptor, FcεRI. The in vivo effects of TGFβ1 and the means by which it suppresses mast cells have been less clear. The current study shows that TGFβ1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGFβ1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGFβ1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGFβ1, since Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward SCF, and that TGFβ1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGFβ1 greatly reduced mast cell numbers in Th1-prone C57BL/6 but not Th2-prone 129/Sv mice. Furthermore, TGFβ1 did not suppress IgE-induced cytokine release, and increased c-Kit-mediated migration in 129/Sv mast cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGFβ1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGFβ1-mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGFβ1 regulates mast cell homeostasis, and that this feedback suppression may be dependent upon genetic context, predisposing some individuals to atopic disease.
doi:10.4049/jimmunol.1202723
PMCID: PMC3846427  PMID: 24068671
asthma; allergy; Stat5; Fyn; inflammation; IgE
6.  K63-linked polyubiquitylation of IRF1 transcription factor is essential for IL-1-induced CCL5 and CXCL10 chemokine production 
Nature immunology  2014;15(3):231-238.
Although interleukin-1 (IL-1) induces expression of interferon regulatory factor 1 (IRF1), its roles in immune and inflammatory responses and mechanisms of activation remain elusive. Here, we show that IRF1 is essential for IL-1-induced expression of chemokines CXCL10 and CCL5 that recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquires K63-linked polyubiquitylation mediated by cellular inhibitor of apoptosis 2 (cIAP2), which is enhanced by the bioactive lipid sphingosine-1 phosphate (S1P). In response to IL-1, cIAP2 and sphingosine kinase 1, the enzyme that generates S1P, form a complex with IRF1, which leads to its activation. Thus, IL-1 triggers a hitherto unknown signaling cascade that controls induction of IRF1-dependent genes important for sterile inflammation.
doi:10.1038/ni.2810
PMCID: PMC3976678  PMID: 24464131
7.  Resection of the Primary Tumor Improves Survival in Metastatic Breast Cancer by Reduction of Overall Tumor Burden 
Surgery  2013;153(6):771-778.
Background
Although many retrospective studies suggest resection of the primary tumor improves survival in metastatic breast cancer, animal studies suggest resection induces metastasis. Moreover, there has been no critical evaluation of how well animal studies actually model metastatic breast cancer. We utilized our newly established orthotopic cancer implantation under direct vision model to evaluate the hypothesis that primary tumor resection improves survival in metastatic breast cancer by reduction of overall tumor burden and improved immune responsiveness.
Methods
Murine mammary adenocarcinoma 4T1-luc2 cells that can be visualized by bioluminescence were implanted orthotopically into Balb/c mice under direct vision. Resection of the primary tumors at Days 6, 10, and 28 were compared to sham resection of the contralateral normal mammary gland and observation alone. Tumor burden was quantified by bioluminescence. Tumor draining lymph nodes were identified by intradermal injection of lymphazurin, and primary tumors, lymph nodes and lungs were examined pathologically. Kaplan-Meier survival analyses were performed. Splenocyte myeloid derived suppressor cells (MDSCs) and CD4 or CD8 single positive T lymphocytes were quantified by flow cytometry.
Results
Tumors invaded locally, metastasized to regional lymph nodes, and then to distant organs, with subsequent mortality. Surgical stress increased tumor burden only transiently without affecting survival. When primary tumor resection decreased overall tumor burden substantially, further growth of metastatic lesions did not increase overall tumor burden compared to observation and survival was improved, which was not the case when resection did not significantly reduce overall tumor burden. Decreasing overall tumor burden through resection of the primary tumor resulted in decreased splenic MDSC numbers and increased CD4 and CD8 cells, suggesting the potential for an improved immunological response against cancer.
Conclusions
Decreasing overall tumor burden through resection of the primary breast tumor decreased MDSCs, increased CD4 and CD8 cells, and improved survival.
doi:10.1016/j.surg.2013.02.002
PMCID: PMC3664113  PMID: 23489938
8.  Sphingosine kinase: a closer look at last 
Sphingosine-1-phosphate is a potent sphingolipid mediator and the kinase that produces it, sphingosine kinase 1 (SphK1), has been implicated in cancer progression, inflammation, and cardiovascular diseases. In this issue of Structure, Wang and colleagues provide the scientific community with the long awaited structure of SphK1 (Wang et al., 2013).
doi:10.1016/j.str.2013.04.006
PMCID: PMC3711075  PMID: 23664359
9.  Memo Has a Novel Role in S1P Signaling and Crucial for Vascular Development 
PLoS ONE  2014;9(4):e94114.
Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.
doi:10.1371/journal.pone.0094114
PMCID: PMC3979765  PMID: 24714781
10.  A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell-dependent mouse model of allergic asthma 
Background
Sphingosine-1-phosphate (S1P) produced by two sphingosine kinase isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution and the role that S1P plays in vivo in a mast cell and IgE-dependent mouse model of allergic asthma has not yet been examined.
Objective
We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.
Methods
Allergic airway inflammation and AHR were examined in a mast cell-dependent mouse model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I prior to sensitization and challenge with OVA or only prior to challenge.
Results
SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of NF-κB, a master transcription factor that regulates expression of pro-inflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, which develop mast cell-dependent allergic inflammation, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, 5, 6, 13, IFN-γ, and TNF-α and the chemokines eotaxin, and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation as well as activation of NF-κB in the lungs.
CONCLUSION
S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.
doi:10.1016/j.jaci.2012.07.014
PMCID: PMC3563730  PMID: 22939756
sphingosine-1-phosphate; sphingosine kinase; mast cells; NF-kB; airway hyperresponsiveness; asthma
11.  Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond 
Nature reviews. Drug discovery  2013;12(9):688-702.
The bioactive lipid sphingosine-1-phosphate (S1P) is involved in multiple cellular signalling systems and has a pivotal role in the control of immune cell trafficking. As such, S1P has been implicated in disorders such as cancer and inflammatory diseases. This Review discusses the ways in which S1P might be therapeutically targeted — for example, via the development of chemical inhibitors that target the generation, transport and degradation of S1P and via the development of specific S1P receptor agonists. We also highlight recent conflicting results observed in preclinical studies targeting S1P and discuss ongoing clinical trials in this field.
doi:10.1038/nrd4099
PMCID: PMC3908769  PMID: 23954895
12.  Sphingosine-1-Phosphate Links Persistent STAT3 Activation, Chronic Intestinal Inflammation, and Development of Colitis-Associated Cancer 
Cancer cell  2012;23(1):107-120.
SUMMARY
Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The pro-drug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC even in Sphk2−/− mice and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.
doi:10.1016/j.ccr.2012.11.013
PMCID: PMC3578577  PMID: 23273921
13.  Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy 
Disease Models & Mechanisms  2013;7(1):41-54.
Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.
doi:10.1242/dmm.013631
PMCID: PMC3882047  PMID: 24077965
HDAC; S1P; THI; dys; Dystrophin; mdx
14.  Is tail vein injection a relevant breast cancer lung metastasis model? 
Journal of Thoracic Disease  2013;5(4):385-392.
Background
Two most commonly used animal models for studying breast cancer lung metastasis are: lung metastasis after orthotopic implantation of cells into the mammary gland, and lung implantations produced after tail vein (TV) injection of cells. Tail vein injection can produce lung lesions faster, but little has been studied regarding the differences between these tumors, thus, we examined their morphology and gene expression profiles.
Methods
Syngeneic murine mammary adenocarcinoma, 4T1-luc2 cells, were implanted either subcutaneously (Sq), orthotopically (OS), or injected via TV in Balb/c mice. Genome-wide microarray analyses of cultured 4T1 cells, Sq tumor, OS tumor, lung metastases after OS (LMet), and lung tumors after TV (TVt) were performed 10 days after implantation.
Results
Bioluminescence analysis demonstrated different morphology of metastases between LMet and TVt, confirmed by histology. Gene expression profile of cells were significantly different from tumors, OS, Sq, TVt or LMet (10,350 probe sets; FDR≤1%; P<0.0001). Sq tumors were significantly different than OS tumors (700 probe sets; FDR≤15%; P<0.01), and both tumor types (Sq and OS) were significantly different than LMet (1,247 probe sets; >1.5-fold-change; P<0.01), with no significant difference between TVt and LMet.
Conclusions
There were significant differences between the gene profiles of cells in culture and OS versus LMet, but there were no differences between LMet versus TVt. Therefore, the lung tumor generated by TVt can be considered genetically similar to those produced after OS, and thus TVt is a relevant model for breast cancer lung metastasis.
doi:10.3978/j.issn.2072-1439.2013.06.17
PMCID: PMC3755653  PMID: 23991292
Breast cancer; lung metastasis; animal model; microarray; metastasis model
15.  Sorafenib and pemetrexed toxicity in cancer cells is mediated via SRC-ERK signaling 
Cancer Biology & Therapy  2012;13(9):793-803.
The present studies sought to further understand how the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib interact to kill tumor cells. Sorafenib activated SRC, and via SRC the drug combination activated ERK1/2. Expression of dominant negative SRC or dominant negative MEK1 abolished drug-induced ERK1/2 activation, together with drug-induced autophagy, acidic lysosome formation, and tumor cell killing. Protein phosphatase 2A is an important regulator of the ERK1/2 pathway. Fulvestrant resistant MCF7 cells expressed higher levels of the PP2A inhibitor SET/I2PP2A, had lower endogenous PP2A activity, and had elevated basal ERK1/2 activity compared with their estrogen dependent counterparts. Overexpression of I2PP2A blocked drug-induced activation of ERK1/2 and tumor cell killing. PP2A can be directly activated by ceramide and SET/I2PP2A can be inhibited by ceramide. Inhibition of the de novo ceramide synthase pathway blocked drug-induced ceramide generation, PP2A activation and tumor cell killing. Collectively these findings demonstrate that ERK1/2 plays an essential role downstream of SRC in pemetrexed and sorafenib lethality and that PP2A plays an important role in regulating this process.
doi:10.4161/cbt.20562
PMCID: PMC3679099  PMID: 22673740
ERK; I2PP2A; PP2A; SRC; autophagy; ceramide; pemetrexed; sorafenib
16.  Copper Dependence of Angioproliferation in Pulmonary Arterial Hypertension in Rats and Humans 
Obliteration of the vascular lumen by endothelial cell growth is a hallmark of many forms of severe pulmonary arterial hypertension. Copper plays a significant role in the control of endothelial cell proliferation in cancer and wound-healing. We sought to determine whether angioproliferation in rats with experimental pulmonary arterial hypertension and pulmonary microvascular endothelial cell proliferation in humans depend on the proangiogenic action of copper. A copper-depleted diet prevented, and copper chelation with tetrathiomolybdate reversed, the development of severe experimental pulmonary arterial hypertension. The copper chelation–induced reopening of obliterated vessels was caused by caspase-independent apoptosis, reduced vessel wall cell proliferation, and a normalization of vessel wall structure. No evidence was found for a role of super oxide–1 inhibition or lysyl–oxidase–1 inhibition in the reversal of angioproliferation. Tetrathiomolybdate inhibited the proliferation of human pulmonary microvascular endothelial cells, isolated from explanted lungs from control subjects and patients with pulmonary arterial hypertension. These data suggest that the inhibition of endothelial cell proliferation by a copper-restricting strategy could be explored as a new therapeutic approach in pulmonary arterial hypertension. It remains to be determined, however, whether potential toxicity to the right ventricle is offset by the beneficial pulmonary vascular effects of antiangiogenic treatment in patients with pulmonary arterial hypertension.
doi:10.1165/rcmb.2011-0296OC
PMCID: PMC3361355  PMID: 22162909
pulmonary hypertension; copper; angiogenesis; tetrathiomolybdate
17.  Lyn but not Fyn kinase controls IgG-Mediated Systemic Anaphylaxis 
Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage and neutrophil secretion of platelet activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38 and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR–mediated activation was enhanced in Lyn-deficient (KO) cells, but decreased in Fyn KO cells, compared to wild type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features while no change was observed for Fyn KO mice, compared to wild type littermates. Intriguingly, we establish that mast cells account for the majority of serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.
doi:10.4049/jimmunol.1003223
PMCID: PMC3536057  PMID: 22450804
18.  Structure of the First Sphingosine 1-Phosphate Receptor 
Science signaling  2012;5(225):pe23.
The sphingosine 1-phosphate receptor 1 (S1P1) and its ligand, sphingosine 1-phosphate (S1P), have now emerged as critical regulators of lymphocyte trafficking, vascular development and integrity, and immunity. S1P1 is targeted by the phosphorylation product of fingolimod, which has been approved for the treatment of multiple sclerosis. The recent progress in the structural biology of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors has now enabled the elucidation of the structure of S1P1. Analysis of the structure, along with structure activity and mutagenesis analysis, highlighted key interactions associated with the binding of S1P and agonists and suggested that the ligand may gain access to the binding pocket by lateral diffusion within the plasma membrane. The S1P1 crystal structure will be helpful for designing ligands that specifically target S1P1.
doi:10.1126/scisignal.2003160
PMCID: PMC3632326  PMID: 22623751
19.  Biological Characterization of 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) as a Selective Sphingosine Kinase-2 Inhibitor and Anticancer Agent 
PLoS ONE  2013;8(2):e56471.
In our effort to develop selective sphingosine kinase-2 (SphK2) inhibitors as pharmacological tools, a thiazolidine-2,4-dione analogue, 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145), was synthesized and biologically characterized. Biochemical assay results indicate that K145 is a selective SphK2 inhibitor. Molecular modeling studies also support this notion. In vitro studies using human leukemia U937 cells demonstrated that K145 accumulates in U937 cells, suppresses the S1P level, and inhibits SphK2. K145 also exhibited inhibitory effects on the growth of U937 cells as well as apoptotic effects in U937 cells, and that these effects may be through the inhibition of down-stream ERK and Akt signaling pathways. K145 also significantly inhibited the growth of U937 tumors in nude mice by both intraperitoneal and oral administration, thus demonstrating its in vivo efficacy as a potential lead anticancer agent. The antitumor activity of K145 was also confirmed in a syngeneic mouse model by implanting murine breast cancer JC cells in BALB/c mice. Collectively, these results strongly encourage further optimization of K145 as a novel lead compound for development of more potent and selective SphK2 inhibitors.
doi:10.1371/journal.pone.0056471
PMCID: PMC3577900  PMID: 23437140
20.  Endogenous Modulators and Pharmacological Inhibitors of Histone Deacetylases in Cancer Therapy 
Oncogene  2011;31(5):537-551.
The class I histone deacetylases HDAC1 and HDAC2 belong to a family of 11 zinc-dependent human HDACs and are overexpressed in many cancers. Inhibitors of these HDACs now in clinical trials show activity against several types of cancers. This review is focuse on recent advances in both clinical and preclinical efforts to understand the basis for HDACi actions, with an emphasis on implications for rational combinations with conventional or other targeted agents. We will address new perspectives on the molecular mechanisms by which HDACs act and how these actions relate to cancer. We will also review new evidence demonstrating that HDACs are direct intracellular targets of the potent sphingolipid mediator sphingosine-1-phosphate (S1P), the first identified endogenous nuclear regulator of these enzymes, linking sphingolipid metabolism in the nucleus to remodeling of chromatin and epigenetic regulation of gene expression. Understanding how endogenous molecules regulate HDAC activity in vivo may facilitate the search for safer and more effective anti-cancer drugs capable of interfering with HDAC functions in a highly specific manner.
doi:10.1038/onc.2011.267
PMCID: PMC3356932  PMID: 21725353
histone deacetylase; histone deacetylase inhibitor; apoptosis; sphingosine-1-phosphate; cancer
21.  Sphingosine-1-phosphate produced by sphingosine kinase 1 promotes breast cancer progression by stimulating angiogenesis and lymphangiogenesis 
Cancer Research  2012;72(3):726-735.
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacological evidence of a critical role for sphingosine kinase 1 (SphK1) in producing S1P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S1P levels increased both in the tumor and the circulation. In agreement, serum S1P levels were significantly elevated in stage IIIA human breast cancer patients, compared to age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK1 inhibitor SK1-I suppressed S1P levels, reduced metastases to lymph nodes and lungs and decreased overall tumor burden of our murine model. Both S1P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro whereas SK1-I inhibited each process. We quantified both processes in vivo from the same specimen by combining Directed In Vivo Angiogenesis Assays (DIVAA) with Fluorescence Activated Cell Sorting (DIVAA/FACS), thereby confirming the results obtained in vitro. Notably, SK1-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK1-I-treated animals. Taken together, our findings demonstrate that SphK1-produced S1P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK1 along with S1P as therapeutic targets in breast cancer.
doi:10.1158/0008-5472.CAN-11-2167
PMCID: PMC3289261  PMID: 22298596
sphingosine kinase 1; sphingosine-1-phosphate; lymphangiogenesis; angiogenesis; lymph node metastasis
22.  Fenretinide Causes Emphysema, Which Is Prevented by Sphingosine 1-Phoshate 
PLoS ONE  2013;8(1):e53927.
Sphingolipids play a role in the development of emphysema and ceramide levels are increased in experimental models of emphysema; however, the mechanisms of ceramide-related pulmonary emphysema are not fully understood. Here we examine mechanisms of ceramide-induced pulmonary emphysema. Male Sprague-Dawley rats were treated with fenretinide (20 mg/kg BW), a synthetic derivative of retinoic acid that causes the formation of ceramide, and we postulated that the effects of fenretinide could be offset by administering sphingosine 1-phosphate (S1P) (100 µg/kg BW). Lung tissues were analyzed and mean alveolar airspace area, total length of the alveolar perimeter and the number of caspase-3 positive cells were measured. Hypoxia-inducible factor alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other related proteins were analyzed by Western blot analysis. Immunohistochemical analysis of HIF-1α was also performed. Ceramide, dihydroceramide, S1P, and dihydro-S1P were measured by mass spectrometer. Chronic intraperitoneal injection of fenretinide increased the alveolar airspace surface area and increased the number of caspase-3 positive cells in rat lungs. Fenretinide also suppressed HIF-1α and VEGF protein expression in rat lungs. Concomitant injection of S1P prevented the decrease in the expression of HIF-1α, VEGF, histone deacetylase 2 (HDAC2), and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein expression in the lungs. S1P injection also increased phosphorylated sphingosine kinase 1. Dihydroceramide was significantly increased by fenretinide injection and S1P treatment prevented the increase in dihydroceramide levels in rat lungs. These data support the concept that increased de novo ceramide production causes alveolar septal cell apoptosis and causes emphysema via suppressing HIF-1α. Concomitant treatment with S1P normalizes the ceramide-S1P balance in the rat lungs and increases HIF-1α protein expression via activation of sphingosine kinase 1; as a consequence, S1P salvages fenretinide induced emphysema in rat lungs.
doi:10.1371/journal.pone.0053927
PMCID: PMC3543313  PMID: 23326540
23.  Conjugated Bile Acids Activate the Sphingosine-1-Phosphate Receptor 2 in Primary Rodent Hepatocytes 
Hepatology (Baltimore, Md.)  2011;55(1):267-276.
Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including the extracellular regulated kinase (ERK)1/2 and AKT as well as the G-protein coupled receptor (GPCR), TGR5/M-BAR. Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be pertussis toxin (PTX) and dominant negative Gαi sensitive in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid activated phylogenetic family, expressed in HEK293 cells, identified sphingosine 1-phosphate receptor 2 (S1P2) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P-induced activation of ERK1/2 and AKT were significantly inhibited by JTE-013, a S1P2 antagonist, in primary rat hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knock down of the expression of S1P2 by a recombinant lentivirus encoding S1P2 shRNA, markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P2 knock out (S1P2−/−) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P2 can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily via S1P2 in primary rodent hepatocytes.
doi:10.1002/hep.24681
PMCID: PMC3245352  PMID: 21932398
G protein coupled receptor; homology modeling; S1P receptor 2 knockout mice; cell signaling
24.  SPHINGOSINE-1-PHOSPHATE SIGNALING AND ITS ROLE IN DISEASE 
Trends in Cell Biology  2011;22(1):50-60.
The bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), is now recognized as a critical regulator of many physiological and pathophysiological processes, including cancer, atherosclerosis, diabetes and osteoporosis. S1P is produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2. Many cells secrete S1P, which can then act in an autocrine or paracrine manner. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. More recently, it was shown that S1P also has important intracellular targets involved in inflammation, cancer and Alzheimer’s disease. This suggests that S1P actions are much more complex than previously thought, with important ramifications for development of therapeutics. This review highlights recent advances in our understanding of mechanisms of action of S1P and its roles in disease.
doi:10.1016/j.tcb.2011.09.003
PMCID: PMC3253987  PMID: 22001186
25.  DEVELOPMENT OF SMALL-MOLECULE INHIBITORS OF SPHINGOSINE-1-PHOSPHATE SIGNALING 
Pharmacology & therapeutics  2011;132(3):352-360.
The pleiotropic sphingolipid mediator, sphingosine-1-phosphate, produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2, regulates many cellular and physiological processes important for homeostasis and development and pathophysiology. Many of the actions of S1P are mediated by a family of five specific cell surface receptors that are ubiquitously and specifically expressed, although important direct intracellular targets of S1P have also recently been identified. S1P, SphK1, and or S1P receptors have been linked to onset and progression of numerous diseases, including many types of cancer, and especially inflammatory disorders, such as multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease, and sepsis. S1P formation and signaling are attractive targets for development of new therapeutics. The effects of a number of inhibitors of SphKs and S1PRs have been examined in animal models of human diseases. The effectiveness of the immunosuppressant FTY720 (known as Fingolomod or Gilenya), recently approved for the treatment of multiple sclerosis, whose actions are mediated by downregulation of S1PR1, has become the gold standard for S1P-centric drugs. Here, we review S1P biology and signaling with an emphasis on potential therapeutic benefits of specific interventions and discuss recent development of small molecule antagonists and agonists that target specific subtypes of S1P receptors as well as inhibitors of SphKs.
doi:10.1016/j.pharmthera.2011.08.004
PMCID: PMC3205210  PMID: 21906625
sphingosine-1-phosphate; sphingosine kinase; inhibitors; antagonists; cancer; multiple sclerosis; asthma; rheumatoid arthritis; inflammatory bowel disease; sepsis; apoptosis; S1P receptors

Results 1-25 (72)