Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to sphingosine-1-phosphate (S1P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, sphingosine, and S1P. The ubiquitous mediator S1P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca(2+) homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK1 and SphK2 are specifically regulated are complex and only partially understood. Although SphK1 and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK1 or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.
Most drugs currently used for human therapy interact with proteins, altering their activity to modulate the pathological cell physiology. In contrast, 2-hydroxy-9-cis-octadecenoic acid (Minerval) was designed to modify the lipid organization of the membrane. Its structure was deduced following the guidelines of the mechanism of action previously proposed by us for certain antitumor drugs. The antiproliferative activity of Minerval supports the above-mentioned hypothesis. This molecule augments the propensity of membrane lipids to organize into nonlamellar (hexagonal H II ) phases, promoting the subsequent recruitment of protein kinase C (PKC) to the cell membrane. The binding of the enzyme to membranes was marked and significantly elevated by Minerval in model (liposomes) and cell (A549) membranes and in heart membranes from animals treated with this drug. In addition, Minerval induced increased PKC␣ expression (mRNA and protein levels) in A549 cells. This drug also induced PKC activation, which led to a p53-independent increase in p21 CIP expression, followed by a decrease in the cellular concentrations of cyclins A, B, and D3 and cdk2. These molecular changes impaired the cell cycle progression of A549 cells. At the cellular and physiological level, administration of Minerval inhibited the growth of cancer cells and exerted antitumor effects in animal models of cancer without apparent histological toxicity. The present results support the potential use of Minerval and related compounds in the treatment of tumor pathologies.
Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the G i proteincoupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF 4 Ϫ also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca 2؉ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through G i -type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.During the last few years, it has become clear that sphingolipids, in addition to being structural constituents of cell membranes, are sources of important signaling molecules. Particularly, the sphingolipid metabolites, ceramide and sphingosine-1-phosphate (SPP), 1 have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (1-4). Interest in SPP focused recently on two distinct cellular actions of this lipid, namely its function as extracellular ligand activating specific G protein-coupled membrane receptors and its role as intracellular second messenger (5). Important clues to a specific intracellular action of SPP were the following findings. First, activation of various plasma membrane receptors, such as the platelet-derived growth factor receptor (6, 7), the Fc⑀RI (8), and the Fc␥RI antigen receptors (9), was found to rapidly increase intracellular SPP production through stimulation of sphingosine kinase. Second, inhibition of sphingosine kinase stimulation strongly reduced or even prevented cellular events triggered by these tyrosine kinase-linked receptors, such as receptor-stimulated DNA synthesis, Ca 2ϩ mobilization, and vesicular trafficking (6,8,9). Finally, intracellular SPP was found to mimic the receptor responses, i.e. it stimulated DNA synthesis and mobilized Ca 2ϩ from internal stores (10 -14). We recently reported that the G protein-coupled muscarinic acetylcholine receptor subtypes m2 and m3 expressed in HEK-293 cells also induce a rapid and transient SPP production by sphingosine kinase. Furthermore, intracellular in...
We have recently reported that lipid structure regulates the interaction with membranes, recruitment to membranes, and distribution to membrane domains of heterotrimeric G␣␥ proteins, G␣ subunits, and G␥ dimers (J Biol Chem 279: 36540 -36545, 2004). Here, we demonstrate that modulation of the membrane structure not only determines G protein localization but also regulates the function of G proteins and related signaling proteins. In this context, the antitumor drug daunorubicin (daunomycin) and oleic acid changed the membrane structure and inhibited G protein activity in biological membranes. They also induced marked changes in the activity of the ␣ 2A/Dadrenergic receptor and adenylyl cyclase. In contrast, elaidic and stearic acid did not change the activity of the abovementioned proteins. These fatty acids are chemical but not structural analogs of oleic acid, supporting the structural basis of the modulation of membrane lipid organization and subsequent regulation of G protein-coupled receptor signaling. In addition, oleic acid (and also daunorubicin) did not alter G protein activity in a membrane-free system, further demonstrating the involvement of membrane structure in this signal modulation. The present work also unravels in part the molecular bases involved in the antihypertensive (Hypertension 43: 249 -254, 2004) and anticancer (Mol Pharmacol 67:531-540, 2005) activities of synthetic oleic acid derivatives (e.g., 2-hydroxyoleic acid) as well as the molecular bases of the effects of diet fats on human health.
1 The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (western blotting) in the rat brain after treatment with various imidazoline drugs and other agents. 2 Chronic (7 days) but not acute (1 day) treatment with the imidazoline drugs, cirazoline (1 mg kg-', i.p.) and idazoxan (1Omg kg', i.p.), but not with the structurally related a2-adrenoceptor antagonists, RX821002 (2-methoxy idazoxan) (1Omg kg', i.p.) and efaroxan (1O mg kg', i.p.), markedly increased (45%) GFAP immunoreactivity in the rat cerebral cortex. Chronic treatment (7 days) with yohimbine (10mg kg-', i.p.), a non-imidazoline a2-adrenoceptor antagonist, did not significantly modify GFAP immunoreactivity in the cerebral cortex. 3 Chronic treatment (7 days) with cirazoline and idazoxan did not alter the density of brain monoamine oxidase (MAO)-B sites labelled by [3H]-Ro 19-6327 (lazabemide), another relevant astroglial marker. Moreover, these imidazoline drug treatments did not modify the levels of a-tubulin in the cerebral cortex. These negative results reinforced the specificity of the effects of imidazoline drugs on GFAP. 4 Irreversible inactivation of brain x2-adrenoceptors (and other neurotransmitters receptors) after treatment with an optimal dose of the peptide-coupling agent EEDQ (1.6 mg kg-', i.p., for 6-24 h) did not alter GFAP immunoreactivity in the cerebral cortex. These results further disproved the involvement of these receptors on astroglial cells in the tonic control of GFAP levels. 5 The binding of [3H]-idazoxan in the presence of 10-6 M (-)-adrenaline was used to quantitate in parallel 12-imidazoline preferring sites in the rat brain after the same treatments. Chronic treatment (7 days) with cirazoline and idazoxan, but not with RX821002, efaroxan or yohimbine, significantly increased (25%) the density of I2-sites in the cerebral cortex. The up-regulation of I2-sites induced by cirazoline was not observed in the liver, a tissue that also expresses 12-sites but lacks glial cells. 6 A strong correlation (r = 0.97) was found when the mean percentage changes in GFAP immunoreactivity were related to the mean percentage changes in 12 imidazoline sites after the various drug treatments. 7 Together the results suggest a direct physiological function of glial I2-imidazoline preferring sites in the regulation of GFAP levels.
Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.
2 Chronic treatment (7 days) with the I2-selective imidazol(in)e drugs idazoxan (10 mg kg-'), cirazoline (1 mg kg-') and LSL 60101 (10 mg kg-') differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30-kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45-kDa protein only by cirazoline (44%) and those of the 66-kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2-sites (32-42%). 3 Chronic treatment (7 days) with efaroxan (10 mg kg-'), RX821002 (10 mg kg-') and yohimbine (10 mg kg-'), which possess very low affinity for 12-imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2-sites. 4 Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg-') and phenelzine (10 mg kg-') decreased the immunoreactivity of the 29/30-kDa (17-24%), and 66-kDa (23-31%) imidazoline receptor proteins. The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.6 mg kg-', 6 h) also decreased the levels of the three imidazoline receptor proteins (20-47%). These drug treatments consistently decreased the density of I2-sites (31-57%). 5
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