Essential requirement for sphingosine kinase 2 in a sphingolipid apoptosis pathway activated by FTY720 analogues

J Biol Chem. 2007 May 25;282(21):15833-42. doi: 10.1074/jbc.M609124200. Epub 2007 Mar 30.

Abstract

The clinical immunosuppressant FTY720 is a sphingosine analogue that, once phosphorylated by sphingosine kinase 2 (Sphk2), is an agonist of multiple receptor subtypes for sphingosine 1-phosphate. Short exposures to FTY720 afford long term protection in lymphoproliferative and autoimmune disease models, presumably by inducing apoptosis in subsets of cells essential for pathogenesis. Sphingosine itself is pro-apoptotic, and apoptosis induced with FTY720 or sphingosine is thought to proceed independently of their phosphorylation. Following chemical mutagenesis of Jurkat cells we isolated mutants that are selectively resistant to FTY720 analogue AAL(R), as well as natural sphingolipid bases, including sphingosine. Cells lacking functional Sphk2 were resistant to apoptosis induced with AAL(R), indicating that apoptosis proceeds through AAL(R) phosphorylation. Phosphorylation of AAL(R) was also required for induction of lymphocyte apoptosis in mice, as apoptosis was not induced with the non-phosphorylatable chiral analogue, AAL(S). Apoptosis was induced in the spleen but not the thymus of mice administered 1 mg/kg AAL(R), correlating with levels of AAL(R)-phosphate (AFD(R)) in organ extracts. AFD(R) did not induce apoptosis when added to the cell culture medium, indicating that it induces apoptosis through an intracellular target. NBD-labeled AAL(R) localized to the endoplasmic reticulum, and AAL(R) treatment resulted in elevated cytosolic calcium, Bax redistribution from cytosol to mitochondrial and endoplasmic reticulum membranes, and caspase-independent mitochondrial permeabilization in Jurkat cells. We therefore describe an apoptotic pathway triggered by intracellular accumulation of sphingolipid base phosphates and suggest that sphingoid base substrates for Sphk2 acting intracellularly could be useful in the treatment of lymphoproliferative diseases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Apoptosis / genetics
  • Autoimmune Diseases / drug therapy
  • Autoimmune Diseases / enzymology
  • Autoimmune Diseases / genetics
  • Calcium Signaling / drug effects*
  • Calcium Signaling / genetics
  • Caspases / metabolism
  • Cell Membrane Permeability / drug effects
  • Cell Membrane Permeability / genetics
  • Drug Resistance, Neoplasm / drug effects
  • Endoplasmic Reticulum / enzymology
  • Fingolimod Hydrochloride
  • HeLa Cells
  • Humans
  • Immunosuppressive Agents / pharmacology*
  • Jurkat Cells
  • Lymphoproliferative Disorders / drug therapy
  • Lymphoproliferative Disorders / enzymology*
  • Lymphoproliferative Disorders / genetics
  • Mice
  • Mice, Knockout
  • Mitochondria
  • Mutagenesis
  • Organ Specificity / drug effects
  • Phosphorylation / drug effects
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Propylene Glycols / pharmacology*
  • Protein Transport / drug effects
  • Protein Transport / genetics
  • Receptors, Lysosphingolipid / antagonists & inhibitors
  • Sphingolipids / metabolism
  • Sphingolipids / pharmacology
  • Sphingosine / analogs & derivatives*
  • Sphingosine / pharmacology
  • Spleen / enzymology
  • Thymus Gland / enzymology
  • bcl-2-Associated X Protein / metabolism

Substances

  • Immunosuppressive Agents
  • Propylene Glycols
  • Receptors, Lysosphingolipid
  • Sphingolipids
  • bcl-2-Associated X Protein
  • Phosphotransferases (Alcohol Group Acceptor)
  • sphingosine kinase
  • Caspases
  • Fingolimod Hydrochloride
  • Sphingosine