Megan M. Price

Sphingosine-1-Phosphate Links Persistent STAT3 Activation, Chronic Intestinal Inflammation, and Development of Colitis-Associated Cancer

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 prodrug 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.

Sphingosine-1-phosphate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration

The potent lipid mediator sphingosine‐1‐phosphate (S1P) regulates diverse physiological processes by binding to 5 specific GPCRs, although it also has intracellular targets. Here, we demonstrate that S1P, produced in the mitochondria mainly by sphin‐gosine kinase 2 (SphK2), binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. In contrast, S1P did not bind to the closely related protein PHB1, which forms large, multimeric complexes with PHB2. In mitochondria from SphK2‐null mice, a new aberrant band of cytochrome‐c oxidase was detected by blue native PAGE, and interaction between subunit IV of cytochrome‐c oxidase and PHB2 was greatly reduced. Moreover, depletion of SphK2 or PHB2 led to a dysfunction in mitochondrial respiration through cytochrome‐c oxidase. Our data point to a new action of S1P in mitochondria and suggest that interaction of S1P with homomeric PHB2 is important for cytochrome‐c oxidase assembly and mitochondrial respiration.—Strub, G. M., Paillard, M., Liang, J., Gomez, L., Allegood, J. C., Hait, N. C., Maceyka, M., Price, M. M., Chen, Q., Simpson, D. C., Kordula, T., Milstien, S., Lesnefsky, E. J., Spiegel, S. Sphingosine‐1‐phos‐phate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration. FASEB J. 25, 600–612 (2011). www.fasebj.org

Distinct roles of sphingosine kinases 1 and 2 in human mast-cell functions

Sphingosine-1-phosphate (S1P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S1P potently induced degranulation of the human LAD2 mast-cell line and cord blood-derived human mast cells (hMCs). S1P also stimulated production and secretion of cytokines, TNF-alpha and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-1, important modulators of inflammation. S1P is produced in mast cells by the 2 sphingosine kinases, SphK1 and SphK2. SphK1 but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-alpha secretion. Taken together, our data suggest that differential formation of S1P by SphK1 and SphK2 has distinct and important actions in hMCs.

A specific sphingosine kinase 1 inhibitor attenuates airway hyperresponsiveness and inflammation in a mast cell–dependent murine model of allergic asthma

BACKGROUND Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) 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 murine 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 murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. RESULTS SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-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.

K63-linked polyubiquitination of transcription factor IRF1 is essential for IL-1-induced production of chemokines CXCL10 and CCL5

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.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.

Essential roles of sphingosine-1–phosphate receptor 2 in human mast cell activation, anaphylaxis, and pulmonary edema

Allergic disease is endemic in developed nations, and none is more dramatic or deadly than systemic anaphylactic shock (Finkelman, 2007). Unfortunately, this collection of rapid changes to the cutaneous, vasculature, and pulmonary systems may also be the least understood of atopic diseases. A better understanding of the eliciting factors for systemic anaphylaxis is therefore critical for accurate diagnosis and therapeutic intervention. Mast cells, which reside in vascularized tissues and are strategically located at the interfaces of host and environment in skin and mucosal surfaces, are key effectors of IgEmediated allergic disorders, including anaphylaxis, hay fever, eczema, and asthma. Mast cells express FcRI (high-affinity receptors for IgE), which binds antigen (Ag)-specific IgE antibodies, and subsequent exposure to Ag initiates cross-linking and aggregation of FcRI. This cascade of events triggers mast cell activation and secretion of a wide array of inflammatory mediators, such as histamine and other preformed mediators, bioactive lipids, including eicosanoids and platelet-activating factor (PAF), and numerous proinflammatory cytokines and chemokines (Kalesnikoff and Galli, 2008), which are essential to the pathogenesis of allergic diseases and anaphylaxis (Finkelman, 2007). A recent addition to the recognized repertoire of lipid mediators secreted by mast cells is the sphingolipid metabolite sphingosine1–phosphate (S1P; Jolly et al., 2004; Mitra et al., 2006; Olivera et al., 2006), which has been implicated in initiation and maintenance of diverse aspects of immune cell activation and function (Rosen et al., 2008; for review see Schwab and Cyster, 2007; Rivera et al., 2008). Focus on S1P in immune responses has increased after the elucidation of its critical role in lymphocyte trafficking (Rosen et al., 2008; for review see Schwab and Cyster, 2007), and observations of local increases of S1P in inflammatory disorders such as asthma (Ammit et al., 2001) and rheumatoid arthritis (Kitano et al., 2006). S1P is a ligand for five G protein–coupled receptors, designated S1P1-5, through which it exerts many of its actions (Spiegel and Milstien, 2003). CORRESPONDENCE Carole Oskeritzian: coskerit@vcu.edu

Sphingosine-1-phosphate induces development of functionally mature chymase-expressing human mast cells from hematopoietic progenitors

Mast cells (MCs) play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow‐derived progenitors that circulate in the blood as immature precursors. MCs developed from cord blood‐derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCTs), the phenotype predominant in the lung. MC progenitors are likely to encounter the serum‐borne bioactive sphingolipid metabolite, sphingosine‐1‐phosphate (S1P), during migration to target tissues. S1P accelerated the development of cord blood‐derived MCs (CB‐MCs) and strikingly increased the numbers of MC‐expressing chymase. These MCs have functional FcsRIs, and similar to skin MCTCs that express both tryptase and chymase, also express CD88 and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL‐6, a cytokine known to promote development of functionally mature MCTCs, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage‐depleted CB‐MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP‐1/CCL2), from these macrophage‐depleted and purified CB‐MCs. These results suggest crucial roles for S1P in regulating development of human MCs and their functions and reveal a complex interplay between macrophages and MC progenitors in the development of mature human MCs.—Price, M. M., Kapitonov, D., Allegood, J., Milstien, S., Oskeritzian, C. A., Spiegel, S. Sphingosine‐1‐phosphate induces development of functionally mature chymase‐expressing human mast cells from hematopoietic progenitors. FASEB J. 23, 3506–3515 (2009). www.fasebj.org

Sphingosine-1-phosphate synthesis and functions in mast cells

Sphingolipids are not only major lipid components of all eukaryotic cell membranes, but they also comprise an important family of bioactive signaling molecules that regulate a diverse array of biological responses. The sphingolipid metabolite sphingosine-1-phosphate (S1P), is a key regulator of immune responses. Cellular levels of S1P are determined by the balance between its synthesis, involving two sphingosine kinases (SphK1 and SphK2), and its degradation, involving S1P lyase and S1P phosphatases. S1P mainly signals through its cell-surface receptors and may also have intracellular functions. S1P has important functions in mast cells - the major effectors of allergic responses. Antigen triggering of IgE receptors on mast cells activates both SphKs resulting in the production of S1P that is released and regulates and amplifies mast cell functions, including degranulation as well as cytokine and chemokine release.

Abstract LB-325: Sphingosine-1-phosphate links chronic intestinal inflammation to development of colitis-associated cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Inflammatory bowel disease is an important risk factor for colorectal cancer. The pro-inflammatory cytokines TNF-alpha and IL-6 and their downstream master transcription factors NF-kappaB and Stat3 are critical regulators of chronic inflammation and cancer. There is growing evidence that sphingosine-1-phosphage (S1P), a pleiotropic bioactive sphingolipid metabolite formed inside cells by two closely related sphingosine kinases, SphK1 and SphK2, is involved in inflammation and cancer. In this work, we have shown that S1P produced by upregulation of SphK1 links chronic intestinal inflammation to CAC and both are exacerbated by deletion of SphK2. S1P is essential for production of the multifunctional NF-kappaB-regulated cytokine IL-6, persistent activation of the transcription factor Stat3, and consequent upregulation of the S1P receptor, S1PR1. Adoptive transfer demonstrated that SphK2 deficiency in hematopoietic cells contributed to colitis severity, IL-6 production, and activation of Stat3 and NF-kappaB. The pro-drug FTY720 decreased SphK1 and S1PR1 and eliminated the NF-kappaB-IL-6-Stat3 amplification cascade and development of CAC even in SphK2-/- mice, suggesting that FTY720 may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1-S1P-S1PR1 axis is at the nexus between NF-kappaB and Stat3 and connects chronic inflammation and CAC. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and K12HD055881 and the Susan G. Komen for the Cure Research Foundation grant (to K.T.). M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. E.Y.K. and J.C.A. were supported by NIH training grant T32HL094290. Citation Format: Masayuki Nagahashi, Jie Liang, Eugene Y. Kim, Kuzhuvelil B. Harikumar, Akimitsu Yamada, Wei-Ching Huang, Nitai C. Hait, Jeremy C. Allegood, Megan M. Price, Dorit Avni, Kazuaki Takabe, Tomasz Kordula, Sheldon Milstien, Sarah Spiegel. Sphingosine-1-phosphate links chronic intestinal inflammation to development of colitis-associated cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-325. doi:10.1158/1538-7445.AM2013-LB-325