Michael Maceyka

Regulation of Histone Acetylation in the Nucleus by Sphingosine-1-Phosphate

Epigenetic Signals The lipid sphingosine-1-phosphate (S1P) is a signaling molecule that binds to receptors on the cell surface to initiate biochemical changes that control a range of biological processes from growth and survival to immune reactions. Hait et al. (p. 1254) report that S1P can also function by direct binding to the nuclear enzymes, histone deacetylases (HDACs) 1 and 2. The enzyme that generates S1P, sphingosine kinase 2 (ShpK2) is present in the nucleus in complexes with HDAC1 and HDAC2. Generation of S1P and its binding to HDACs inhibited deacetylation of histone. Such histone modification is an epigenetic mechanism that controls gene transcription. Thus, generation of S1P in the nucleus appears to be a signaling mechanism by which cells can control gene expression in response to various stimuli. A phospholipid that binds to nuclear enzymes modifies gene transcription in response to external stimuli. The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) can act intracellularly independently of its cell surface receptors through unknown mechanisms. Sphingosine kinase 2 (SphK2), one of the isoenzymes that generates S1P, was associated with histone H3 and produced S1P that regulated histone acetylation. S1P specifically bound to the histone deacetylases HDAC1 and HDAC2 and inhibited their enzymatic activity, preventing the removal of acetyl groups from lysine residues within histone tails. SphK2 associated with HDAC1 and HDAC2 in repressor complexes and was selectively enriched at the promoters of the genes encoding the cyclin-dependent kinase inhibitor p21 or the transcriptional regulator c-fos, where it enhanced local histone H3 acetylation and transcription. Thus, HDACs are direct intracellular targets of S1P and link nuclear S1P to epigenetic regulation of gene expression.

Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signalling triggered by TNF-α. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1) that then serves as a platform for recruitment and stimulation of IκB kinase, leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1), one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IκB kinase and IκBα, and IκBα degradation, leading to NF-κB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-κB activation pathway important in inflammatory, antiapoptotic and immune processes.

Sphingosine-1-phosphate signaling and its role in disease

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 Alzheimers 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 the mechanisms of action of S1P and its roles in disease.

SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism

The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca2+ transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions.

Sphingolipid metabolites in inflammatory disease

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.

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.

Extracellular and Intracellular Actions of Sphingosine-1-Phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator with crucial roles in a wide variety of cellular functions across a broad range of organisms. Though a simple molecule in structure, S1P functions are complex. The formation of S1P is catalyzed by one of two sphingosine kinases that have differential cellular distributions as well as both overlapping and opposing functions and which are activated by many different stimuli. S1P can act on a family of G protein-coupled receptors (S1PRs) that are also differentially expressed in different cell types, which influences the cellular responses to S1P. In addition to acting on receptors located on the plasma membrane, S1P can also function inside the cell, independently of S1PRs. It also appears that both the intracellular location and the isotype of sphingosine kinase involved are major determinants of inside-out signaling of S1P in response to many extracellular stimuli. This chapter is focused on the current literature on extracellular and intracellular actions of S1P.

Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond

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.

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

Sphingosine kinase 1 is required for migration, proliferation and survival of MCF-7 human breast cancer cells

Sphingosine‐1‐phosphate (S1P) is a potent lysolipid involved in a variety of biological responses important for cancer progression. Therefore, we investigated the role of sphingosine kinase type 1 (SphK1), the enzyme that makes S1P, in the motility, growth, and chemoresistance of MCF‐7 breast cancer cells. Epidermal growth factor (EGF), an important growth factor for breast cancer progression, activated and translocated SphK1 to plasma membrane. SphK1 was required for EGF‐directed motility. Downregulation of SphK1 in MCF‐7 cells reduced EGF‐ and serum‐stimulated growth and enhanced sensitivity to doxorubicin, a potent chemotherapeutic agent. These results suggest that SphK1 may be critical for growth, metastasis and chemoresistance of human breast cancers.