Digestive Diseases and Sciences

, Volume 53, Issue 4, pp 997–1012 | Cite as

Suppression of Ulcerative Colitis in Mice by Orally Available Inhibitors of Sphingosine Kinase

  • Lynn W. MainesEmail author
  • Leo R. Fitzpatrick
  • Kevin J. French
  • Yan Zhuang
  • Zuping Xia
  • Staci N. Keller
  • John J. Upson
  • Charles D. Smith
Original Paper


A critical step in the mechanism of action of inflammatory cytokines is the stimulation of sphingolipid metabolism, including activation of sphingosine kinase (SK), which produces the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). We have developed orally bioavailable compounds that effectively inhibit SK activity in vitro in intact cells and in cancer models in vivo. In this study, we assessed the effects of these SK inhibitors on cellular responses to tumor necrosis factor alpha (TNFα) and evaluated their efficacy in the dextran sulfate sodium (DSS) model of ulcerative colitis in mice. Using several cell systems, it was shown that the SK inhibitors block the ability of TNFα to activate nuclear factor kappa B (NFκB), induce expression of adhesion proteins, and promote production of prostaglandin E2 (PGE2). In an acute model of DSS-induced ulcerative colitis, SK inhibitors were equivalent to or more effective than Dipentum in reducing disease progression, colon shortening, and neutrophil infiltration into the colon. The effects of SK inhibitors were associated with decreased colonic levels of inflammatory cytokines TNFα, interleukin (IL)-1β, interferon gamma (IFN)-γ, IL-6, and reduction of S1P levels. A similar reduction in disease progression was provided by SK inhibitors in a chronic model of ulcerative colitis in which the mice received 3-week-long cycles of DSS interspaced with week-long recovery periods. In the chronic model, immunohistochemistry for SK showed increased expression in DSS-treated mice (compared with water-treated controls) that was reduced by drug treatment. S1P levels were also elevated in the DSS group and significantly reduced by drug treatment. Together, these data indicate that SK is a critical component in inflammation and that inhibitors of this enzyme may be useful in treating inflammatory bowel diseases.


Sphingosine kinase Inflammatory bowel disease Ulcerative colitis TNFα 



inflammatory bowel disease


sphingosine kinase


sphingosine 1-phosphate


tumor necrosis factor alpha


prostaglandin E2


nuclear factor kappa B


dextran sulfate sodium




interferon gamma




intracellular adhesion molecule-1


vascular cell adhesion molecule-1


polyethylene glycol


Disease Activity Index




phosphate-buffered saline


liquid chromatography tandem mass spectroscopy



This work was supported by grant 1 R43 DK071395 from the National Institutes of Health.


  1. 1.
    Murthy S, Flanigan A (1999) Animal models of inflammatory bowel disease. In: Morgan DW, Marshall LA (eds): In vivo models of inflammation. Progress in inflammation research. Basel, Birkhauser Verlag, pp 205–236Google Scholar
  2. 2.
    Rask-Madsen J (1998) Soluble mediators and the interaction of drugs in IBD. Drugs Today (Barc.) 34:45–63Google Scholar
  3. 3.
    He S (2004) Key role of mast cells and their major secretory products in inflammatory bowel disease. World J Gastroenterol 10(3):309–318PubMedGoogle Scholar
  4. 4.
    Sandborn WJ (2003) Strategies for targeting tumour necrosis factor in IBD. Best Pract Res Clin Gastroenterol 17:105–117PubMedCrossRefGoogle Scholar
  5. 5.
    Loncar M, Al-azzeh ED, Sommer PS, Marinovic M, Schmehl K, Kruschewski M, Blin N, Stohwasser R, Gott P, Kayademir T (2003) Tumour necrosis factor alpha and nuclear factor kappa B inhibits transcription of human TFF3 encoding a gastrointestinal healing peptide. Gut 52:1297–1303PubMedCrossRefGoogle Scholar
  6. 6.
    Van der Woude C, Kleibeuker JH, Jansen PL, Moshage H (2004) Chronic inflammation, apoptosis and (pre-)malignant lesions in the gastro-intestinal tract. Apoptosis 9:123–130PubMedCrossRefGoogle Scholar
  7. 7.
    Singh V, Patil CS, Jain NK, Singh A, Kulkarni SK (2003) Effects of nimesulide on acetic acid- and leukotriene-induced inflammatory bowel disease in rats. Prostaglandins Other Lipid Mediat 71:163–175PubMedCrossRefGoogle Scholar
  8. 8.
    Mahadevan U, Loftus EV, Tremaine WJ, Sandborn WJ (2002) Safety of selective cyclooxygenase-2 inhibitors in inflammatory bowel disease. Am J Gastroenterol 97:910–914PubMedCrossRefGoogle Scholar
  9. 9.
    Kruidenier LKI, Lamers CB, Verspaget HW (2003) Intestinal oxidative damage in inflammatory bowel disease: semi-quantification, localization and association with mucosal antioxidants. J Pathol 201:28–36PubMedCrossRefGoogle Scholar
  10. 10.
    Olivera A, Spiegel S (2001) Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins 64:123–134PubMedGoogle Scholar
  11. 11.
    Spiegel S (1999) Sphingosine 1-phosphate: a prototype of a new class of second messengers. J Leukoc Biol 65:341–344PubMedGoogle Scholar
  12. 12.
    Alessenko AV (2000) The role of sphingomyelin cycle metabolites in transduction of signals of cell proliferation, differentiation and death. Membr Cell Biol 13:303–320PubMedGoogle Scholar
  13. 13.
    Dressler KA, Mathias S, Kolesnick RN (1992) Tumor necrosis factor-a activates the sphingomyelin signal transduction pathway in a cell-free system. Science 255:1715–1718PubMedCrossRefGoogle Scholar
  14. 14.
    Xia P, Gamble JR, Rye KA, Wang L, Hii CS, Cockerill P, Khew-Goodall Y, Bert AG, Barter PJ, Vadas MA (1998) Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway. Proc Natl Acad Sci USA 95:14196–14201PubMedCrossRefGoogle Scholar
  15. 15.
    Mathias S, Pena LA, Kolesnick RN (1998) Signal transduction of stress via ceramide. Biochem J 335(Pt 3):465–480PubMedGoogle Scholar
  16. 16.
    Perry DK, Hannun YA (1998) The role of ceramide in cell signaling. Biochim Biophys Acta 1436:233–243PubMedGoogle Scholar
  17. 17.
    Hayakawa M, Jayadev S, Tsujimoto M, Hannun YA, Ito F (1996) Role of ceramide in stimulation of the transcription of cytosolic phospholipase A2 and cyclooxygenase 2. Biochem Biophys Res Commun 220:681–686PubMedCrossRefGoogle Scholar
  18. 18.
    Gomez-Munoz A, Kong J, Salh B, Steinbrecher UP (2003) Sphingosine 1-phosphate inhibits acid sphingomyelinase and blocks apoptosis in macrophages. FEBS Lett 539:56–60PubMedCrossRefGoogle Scholar
  19. 19.
    Yatomi Y, Ruan F, Hakomori S, Igarashi Y (1995) Sphingosine-1-phosphate: a platelet-activating sphingolipid released from agonist-stimulated human platelets. Blood 86:193–202PubMedGoogle Scholar
  20. 20.
    Prieschl EE, Csonga R, Novotny V, Kikuchi GE, Baumruker T (1999) The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering. J Exp Med 190:1–8PubMedCrossRefGoogle Scholar
  21. 21.
    Pettus BJ, Bielawski J, Porcelli AM, Reames DL, Johnson KR, Morrow J, Chalfant CE, Obeid LM, Hannun YA (2003) The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha. Faseb J 17:1411–1421PubMedCrossRefGoogle Scholar
  22. 22.
    Mackinnon A, Buckley A, Chilvers ER, Rossi AG, Haslet C, Sethi T (2002) Sphingosine kinase: a point of convergence in the action of diverse neutrophil priming agents. J Immunol 169(11):6394–6400PubMedGoogle Scholar
  23. 23.
    Itagaki K, Hauser CJ (2003) Sphingosine 1-phosphate, a diffusible calcium influx factor mediating store-operated calcium entry. J Biol Chem 278:27540–27547PubMedCrossRefGoogle Scholar
  24. 24.
    French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN, Eberly JL, Yun JK, Smith CD (2003) Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res 63:5962–5969PubMedGoogle Scholar
  25. 25.
    French KJ, Upson JJ, Smith SN, Woll M, Zhuang Y, Yun JK, Smith CD (2006) Antitumor activity of sphingosine kinase inhibitors. J Pharmacol Exp Ther 318(2):596–603 PubMedCrossRefGoogle Scholar
  26. 26.
    Maines LW, French KJ, Wolpert EB, Antonetti DA, Smith CD (2006) Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases. Invest Ophth Vis Sci 47:5022–5031CrossRefGoogle Scholar
  27. 27.
    Bohlen P, Stein S, Dairman W, Udenfriend S (1973) Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophys 155:213–220PubMedCrossRefGoogle Scholar
  28. 28.
    Fitzpatrick LR, Wang J, Le M (2000) In vitro and in vivo effects of gliotoxin, a fungal metabolite: efficacy against dextran sodium sulfate-induced colitis in rats. Dig Dis Sci 45:2327–2336PubMedCrossRefGoogle Scholar
  29. 29.
    McCafferty DM, Miampamba M, Sihota E, Sharkey KA, Kubes P (1999) Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice. Gut 45:864–873PubMedCrossRefGoogle Scholar
  30. 30.
    Fiorucci S, Mencarelli A, Palazzetti B, Distrutti E, Vergnolle N, Hollenberg MD, Wallace JL, Morelli A, Cirino G (2001) Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis. Proc Natl Acad Sci USA 98:13936–13941PubMedCrossRefGoogle Scholar
  31. 31.
    Williams KL, Fuller CR, Dieleman LA, DaCosta CM, Haldeman KM, Sartor RB, Lund PK (2001) Enhanced survival and mucosal repair after dextran sodium sulfate-induced colitis in transgenic mice that overexpress growth hormone. Gastroenterology 120:925–937PubMedCrossRefGoogle Scholar
  32. 32.
    Krieglstein CF, Cerwinka WH, Laroux FS, Grisham MB, Schurmann G, Bruwer M, Granger DN (2001) Role of appendix and spleen in experimental colitis. J Surg Res 101:166–175PubMedCrossRefGoogle Scholar
  33. 33.
    Maines LW, Antonetti DA, Wolpert EB, Smith CD (2005) Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazepine by bovine retinal endothelial cells. Neuropharmacology 49:610–617PubMedGoogle Scholar
  34. 34.
    Xia P, Wang L, Gamble JR, Vadas MA (1999) Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells. J Biol Chem 274:34499–34505PubMedCrossRefGoogle Scholar
  35. 35.
    Osawa Y, Banno Y, Nagaki M, Brenner DA, Naiki T, Nozawa Y, Nakashima S, Moriwaki H (2001) TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes. J Immunol 167:173–180PubMedGoogle Scholar
  36. 36.
    Niwa M, Kozawa O, Matsuno H, Kanamori Y, Hara A, Uematsu T (2000) Tumor necrosis factor-alpha-mediated signal transduction in human neutrophils: involvement of sphingomyelin metabolites in the priming effect of TNF-alpha on the fMLP-stimulated superoxide production. Life Sci 66:245–256PubMedCrossRefGoogle Scholar
  37. 37.
    Fueller M, Wang de A, Tigyi G, Siess W (2003) Activation of human monocytic cells by lysophosphatidic acid and sphingosine-1-phosphate. Cell Signal 15:367–375PubMedCrossRefGoogle Scholar
  38. 38.
    Jolly P, Bektas M, Olivera A, Gonzalez-Espinosa C, Proia RL, Rivera J, Milstien S, Spiegel S (2004) Transactivation of sphingosine-1-phospate receptors by Fc{varepsilon}RI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 199:959–970PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Lynn W. Maines
    • 1
    Email author
  • Leo R. Fitzpatrick
    • 2
  • Kevin J. French
    • 1
  • Yan Zhuang
    • 1
  • Zuping Xia
    • 1
  • Staci N. Keller
    • 1
  • John J. Upson
    • 1
  • Charles D. Smith
    • 1
    • 3
  1. 1.Apogee Biotechnology CorporationHersheyUSA
  2. 2.Department of SurgeryPenn State College of MedicineHersheyUSA
  3. 3.Department of PharmacologyPenn State College of MedicineHersheyUSA

Personalised recommendations