Sphingosine-1-phosphate is a potent lipid mediator formed by phosphorylation of sphingosine, a metabolite of sphingolipids, catalyzed by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2. Expression of SphK2, which is enriched in the nucleus of MCF7 human breast cancer cells, increased expression of the cyclin-dependent kinase inhibitor p21 but had no effect on p53 or its phosphorylation. The anticancer drug doxorubicin is known to increase p21 via p53-dependent and p53-independent mechanisms. Down-regulation of endogenous SphK2 with small interfering RNA targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p21 without affecting increased expression of p53. Down-regulation of SphK2 also decreased G 2 -M arrest and markedly enhanced apoptosis induced by doxorubicin. Moreover, siSphK2 reduced doxorubicin-induced p21 expression in p53-inactivated MCF7 cells. Likewise, in human wildtype p53-and p21-expressing HCT116 colon carcinoma cells, as well as in p53-null counterparts, down-regulation of SphK2 markedly reduced p21 induction by doxorubicin. Knockdown of SphK2 sensitized HCT116 cells to apoptosis induced by doxorubicin with concomitant cleavage of poly(ADP-ribose) polymerase. Collectively, our results show that endogenous SphK2 is important for p53-independent induction of p21 expression by doxorubicin and suggest that SphK2 may influence the balance between cytostasis and apoptosis of human cancer cells. [Cancer Res 2007;67(21):10466-74]
The sphingolipid metabolites ceramide and sphingosine-1-phosphate (S1P) have recently been implicated in autophagy. In this study, we report that depletion of sphingosine-1-phosphate phosphohydrolase-1 (SPP1), an endoplasmic reticulum (ER)-resident enzyme that specifically dephosphorylates S1P, induced autophagy. Although the mammalian target of rapamycin and class III phosphoinositide 3-kinase/Beclin-1 pathways were not involved and this autophagy was p53 independent, C/EBP homologous protein, BiP, and phospho-eucaryotic translation initiation factor-2a, and cleavage of procaspases 2 and 4, downstream targets of ER stress, were increased after SPP1 depletion. Autophagy was suppressed by depletion of protein kinase regulated by RNA-like ER kinase (PERK), inositol-requiring transmembrane kinase/endonuclease-1a, or activating transcription factor 6, three sensors of the unfolded protein response (UPR) to ER stress. Autophagy triggered by downregulation of SPP1 did not lead to apoptosis but rather stimulated, in a PERK dependent manner, the survival signal Akt, whose inhibition then sensitized cells to apoptosis. Although depletion of SPP1 increased intracellular levels of S1P and its secretion, activation of cell surface S1P receptors did not induce autophagy. Nevertheless, increases in intracellular pools of S1P, but not dihydro-S1P, induced autophagy and ER stress. Thus, SPP1, by regulating intracellular S1P homeostasis, can control the UPR and ER stress-induced autophagy.
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also upregulated SphK1 expression in other human cancer cells that endogenously express the LPA 1 receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA 1 receptor. An LPA 1 receptor antagonist or down-regulation of its expression prevented SphK1 and S1P 3 receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P 3 expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P 3 , but not S1P 1 , also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells. [Cancer Res 2008;68(16):6569-77]
In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C 6 -ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B 1 , an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.Sphingolipids are a structurally diverse family of membrane lipids. Several lines of evidence have implicated metabolites of sphingolipids such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P) 5 in diverse cellular processes (1, 2). Ceramide and sphingosine have been implicated in pathways involving stress responses, cell differentiation, apoptosis, and cell cycle arrest (1). Unlike ceramide and sphingosine, S1P promotes cell growth and survival and inhibits apoptosis (2, 3). Because of their inter-convertibility and opposing effects, the dynamic balance between S1P and ceramide/sphingosine has been proposed to be an important factor that determines cell fate (2). Accumulating evidence suggests that the S1P/ceramide balance is ultimately regulated by the relative activities of enzymes controlling the turnover of these sphingolipid metabolites. However, the molecular mechanisms involved in the regulation of intracellular levels of these sphingolipids are not yet fully understood. Cellular levels of S1P are kept low by tight spatio-temporal regulation of its synthesis and degradation. Sphingosine kinases (SphKs) catalyze the synthesis of S1P by phosphorylation of sphingosine. Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized in mammals (2). Diverse external stimuli, particularly growth and survival factors, stimulate SphK1, generating S1P that has been implicated in their mitogenic and anti-apoptotic effects (4, 5). In contrast to SphK1, rather than promoting growth and survival, overexpression of SphK2 suppressed growth and...
Background:The switch between autophagy and apoptosis is an important and complicated process that is not well understood. Results: Doxorubicin treatment switches protective autophagy in sphingosine-1-phosphate phosphohydrolase-1 (SPP1)-depleted cells to apoptosis, increasing ceramide synthesis that enhances calpain activation and cleavage of pro-autophagic Atg5, generating a pro-apoptotic fragment. Conclusion: Depletion of SPP1 sensitizes cells to doxorubicin-induced apoptosis. Significance: Sphingolipid metabolites are involved in the cross-talk between autophagy and apoptosis.
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