Nitai C. Hait

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.

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.

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.

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.

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

Estradiol Induces Export of Sphingosine 1-Phosphate from Breast Cancer Cells via ABCC1 and ABCG2

Sphingosine 1-phosphate (S1P), a potent sphingolipid mediator produced by sphingosine kinase isoenzymes (SphK1 and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK1, but not SphK2, increased S1P export from MCF-7 cells. Whereas for both estradiol (E2) and epidermal growth factor-activated SphK1 and production of S1P, only E2 stimulated rapid release of S1P and dihydro-S1P from MCF-7 cells. E2-induced S1P and dihydro-S1P export required estrogen receptor-α, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC1 (multidrug resistant protein 1) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E2-induced activation of ERK1/2, indicating that E2 activates ERK via downstream signaling of S1P. Taken together, our findings suggest that E2-induced export of S1P mediated by ABCC1 and ABCG2 transporters and consequent activation of S1P receptors may contribute to nongenomic signaling of E2 important for breast cancer pathophysiology.

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.

Role of sphingosine kinase 2 in cell migration toward epidermal growth factor

Sphingosine 1-phosphate (S1P), produced by two sphingosine kinase isoenzymes, denoted SphK1 and SphK2, is the ligand for a family of five specific G protein-coupled receptors that regulate cytoskeletal rearrangements and cell motility. Whereas many growth factors stimulate SphK1, much less is known of the regulation of SphK2. Here we report that epidermal growth factor (EGF) stimulated SphK2 in HEK 293 cells. This is the first example of an agonist-dependent regulation of SphK2. Chemotaxis of HEK 293 cells toward EGF was inhibited by N,N-dimethylsphingosine, a competitive inhibitor of both SphKs, implicating S1P generation in this process. Down-regulating expression of SphK1 in HEK 293 cells with a specific siRNA abrogated migration toward EGF, whereas decreasing SphK2 expression had no effect. EGF contributes to the invasiveness of human breast cancer cells, and EGF receptor expression is associated with poor prognosis. EGF also stimulated SphK2 in MDA-MB-453 breast cancer cells. Surprisingly, however, down-regulation of SphK2 in these cells completely eliminated migration toward EGF without affecting fibronectin-induced haptotaxis. Our results suggest that SphK2 plays an important role in migration of MDA-MB-453 cells toward EGF.

Targeting Sphingosine Kinase 1 Inhibits Akt Signaling, Induces Apoptosis, and Suppresses Growth of Human Glioblastoma Cells and Xenografts

Sphingosine-1-phosphate is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce sphingosine-1-phosphate, is up-regulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and nonestablished human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of extracellular signal-regulated kinase 1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the c-Jun-NH(2)-kinase pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced the tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization, and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease.