Sphingolipid Signaling and Pteridine Biosynthesis

National Institute Of Mental Health

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Abstract

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that regulates many vital biological processes, including cell growth, death, and differentiation. In a continuing and highly successful collaboration with Dr. Sarah Spiegel at Virginia Commonwealth University School of Medicine, we are elucidating the mechanisms by which S1P is produced, how its levels are regulated, and how it mediates such diverse actions. Like other important signaling molecules, intracellular levels of S1P are low and regulated by the balance between synthesis and degradation. S1P is produced by phosphorylation of sphingosine catalyzed by sphingosine kinases (SphK1 and SphK2) after activation by diverse types of external stimuli, dephosphorylated by S1P phosphatases and degraded by a lyase. S1P is a ligand for five specific G protein-coupled receptors (named S1P{sub(1-5)}) that signal through various G proteins to regulate many vital cellular processes and together with its intracellular effects on cell survival and calcium homeostasis, account for the pleiotropic effects of S1P. In fact, no cell in the body has been found that does not express as least one S1P receptor and S1P has been shown to regulate important cellular functions throughout evolution, from slime moulds, plants, yeast, and flies, to mammals. We previously found that while SphK1 was important for cell growth and survival, SphK2 seemed to inhibit proliferation and promote cell death. In a recent study, we provided some possible explanations for this puzzling observation as we found that SphK1 and SphK2 have different cellular localizations and have opposing roles in the regulation of sphingolipid metabolism. Thus, SphK2 increases biosynthesis of ceramide, which is pro-apoptotic, while SphK1 decreases ceramide. Our results suggest that the location of S1P production dictates its functions. We also discovered that the ability of the growth factor, PDGF, to stimulate cell growth and motility depends not only on stimulation of SphK1 and formation of S1P, but its net effects on these process are also determined by the S1P{sub2} receptor, which normally serves as a negative damper. Our results suggest that complex interplay between the PDGF receptor and S1P receptors determines their functions. We have also now found that the epidermal growth factor (EGF) stimulates SphK2, which is the first example of an agonist-dependent regulation of this isozyme, and that chemotaxis of breast cancer cells toward EGF is dependent on SphK2 expression. Since EGFR levels correlate with poor prognosis of breast cancer and EGF contributes to the invasiveness of human breast cancer, our results suggest that SphK2 and S1P formation may play an important role in breast cancer progression. In a previous study, we reported that mast cell functions and thus allergic responses were regulated by IgE driven activation of SphK1 and formation of S1P, which then transactivated S1P receptors that were important for mast cell secretion of inflammatory mediators and their movement to sites of inflammation. Surprisingly, we found that overexpression of SphK1 in mast cells impaired degranulation as well as migration toward antigen. We showed that this was due to S1P-induced internalization and desensitization of S1P receptors. These results have important implications for mast-cell degranulation, migration of cells, cancer as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream and exposed to S1P there. The bioactive phospholipids, lysophosphatidic acid (LPA) and phosphatidic acid (PA), regulate pivotal processes related to the pathogenesis of cancer. Based on similarity to SphKs, we cloned and characterized a novel lipid kinase, designated acylglycerol kinase (AGK), that phosphorylates monoacylglycerol and diacylglycerol to form LPA and PA, respectively. AGK expression was up-regulated in prostate cancers compared with normal prostate tissues from the same patient. Expression of AGK in PC-3 prostate cancer cells markedly increased formation and secretion of LPA, resulting in transactivation of the EGF receptor culminating in enhanced cell proliferation and migration. Our results indicate that AGK can amplify EGF signaling pathways and may play an important role in the pathophysiology of prostate cancer. Systemic immune abnormalities have no known relevance to brain dysfunction in autism. As markers for neuroinflammation, we determined levels of sensitive indicators of immune activation in CSF from children with autism. Quinolinic acid (P = 0.037) and neopterin (P = 0.003) were decreased, and biopterin (P = 0.040) was elevated, compared to controls. Among cytokines, only TNFR II was elevated. Decreased quinolinic acid and neopterin in CSF are paradoxical and suggest dysmaturation of metabolic pathways and absence of concurrent infection, respectively, in autism. Alternatively, they may be produced by microglia but remain localized and not expressed in CSF.