Dagmar Meyer zu Heringdorf

Sphingosine kinase‐mediated Ca2+ signalling by G‐protein‐coupled receptors

Formation of inositol 1,4,5‐trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G‐protein‐coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC–IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine‐1‐phosphate (SPP), is involved in GPCR‐mediated Ca2+ signalling as proposed for platelet‐derived growth factor and FcϵRI antigen receptors. Inhibition of sphingosine kinase by DL‐threo‐dihydrosphingosine and N,N‐dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK‐293 cells without affecting mAChR‐induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK‐293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK‐293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase–SPP pathway in addition to PLC–IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR‐induced Ca2+ signalling.

Regulation and functional roles of sphingosine kinases

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 Ca2+ 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.

Sphingosylphosphorylcholine-biological functions and mechanisms of action.

Compared to the lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), little information is available regarding the molecular mechanisms of action, metabolism and physiological significance of the related sphingosylphosphorylcholine (SPC). S1P and LPA have recently been established as agonists at several G-protein-coupled receptors of the EDG family, S1P additionally serves an intracellular second messenger function. Several cellular effects of SPC can be explained by low-affinity binding to and activation of S1P-EDG receptors. However, certain cellular and subcellular actions of SPC are not shared by S1P, suggesting that SPC, which has been identified in normal blood plasma, ascites and various tissues, is a lipid mediator in its own right. This concept was corroborated by the recent discovery of specific high-affinity G-protein-coupled SPC receptors. In this article, our present knowledge on cellular actions and biological functions of SPC will be reviewed.

Sphingosine-1-phosphate and sphingosylphosphorylcholine constrict renal and mesenteric microvessels in vitro

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act both intracellularly and at G‐protein‐coupled receptors, some of which were cloned and designated as Edg‐receptors. Sphingolipid‐induced vascular effects were determined in isolated rat mesenteric and intrarenal microvessels. Additionally, sphingolipid‐induced elevations in intracellular Ca2+ concentration were measured in cultured rat aortic smooth muscle cells. SPPC and SPP (0.1–100 μmol l−1) caused concentration‐dependent contraction of mesenteric and intrarenal microvessels (e.g. SPPC in mesenteric microvessels pEC50 5.63±0.17 and Emax 49±3% of noradrenaline), with other sphingolipids being less active. The vasoconstrictor effect of SPPC in mesenteric microvessels was stereospecific (pEC50 D‐erythro‐SPPC 5.69±0.08, L‐threo‐SPPC 5.31±0.06) and inhibited by pretreatment with pertussis toxin (Emax from 44±5 to 19±4%), by chelation of extracellular Ca2+ with EGTA and by nitrendipine (Emax from 40±6 to 6±1 and 29±6%, respectively). Mechanical endothelial denudation or NO synthase inhibition did not alter the SPPC effects, while indomethacin reduced them (Emax from 87±3 to 70±4%). SPP and SPPC caused transient increases in intracellular Ca2+ concentrations in rat aortic smooth muscle cells in a pertussis toxin‐sensitive manner. Our data demonstrate that SPP and SPPC cause vasoconstriction of isolated rat microvessels and increase intracellular Ca2+ concentrations in cultured rat aortic smooth muscle cells. These effects appear to occur via receptors coupled to pertussis toxin‐sensitive G‐proteins. This is the first demonstration of effects of SPP and SPPC on vascular tone and suggests that sphingolipids may be an hitherto unrecognized class of endogenous regulators of vascular tone.

Molecular diversity of sphingolipid signalling

Sphingolipid breakdown products are now being recognized to play a dual role in cellular signalling, acting as intracellular as well as extracellular signalling molecules. Both types of action may even be found with one sphingolipid species. The recent demonstration of G protein‐coupled receptors with high affinity for sphingosine 1‐phosphate and sphingosylphosphorylcholine has been followed by the discovery of several novel sphingolipid actions, such as regulation of heart rate, oxidative burst, neurite retraction or platelet activation. Ligand profiles and concentration‐response relationships suggest the existence of putative sphingolipid receptor subtypes. Against this background, several observations on supposed sphingolipid second messenger actions deserve a new evaluation.

Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors

Recently, a family of G-protein-coupled receptors named endothelial differentiation gene (Edg) receptor family has been identified, which are specifically activated by the two serum lipids, sphingosine-1-phosphate and lysophosphatidic acid. Sphingosine-1-phosphate can also act intracellularly to release Ca2+ from intracellular stores. Since in several cell types, G-protein-coupled lysophosphatidic acid or sphingosine-1-phosphate receptors mobilize Ca2+ in the absence of a measurable phospholipase C stimulation, it was analysed here whether intracellular sphingosine-1-phosphate production was the signalling mechanism used by extracellular sphingosine-1-phosphate for mobilization of stored Ca2+. Sphingosine-1-phosphate and the low affinity sphingosine-1-phosphate receptor agonist, sphingosylphosphorylcholine, induced a rapid, transient and nearly complete pertussis toxin-sensitive Ca2+ mobilization in human embryonic kidney (HEK-293) cells. The G-protein-coupled sphingosine-1-phosphate receptors, Edg-1, Edg-3 and Edg-5, were found to be endogenously expressed in these cells. Most interestingly, sphingosine-1-phosphate and sphingosylphosphorylcholine did not induce a measurable production of inositol-1,4,5-trisphosphate or accumulation of inositol phosphates. Instead, sphingosine-1-phosphate and sphingosylphosphorylcholine induced a rapid and transient increase in production of intracellular sphingosine-1-phosphate with a maximum of about 1.4-fold at 30 s. Stimulation of sphingosine-1-phosphate formation by sphingosine-1-phosphate and sphingosylphosphorylcholine was fully blocked by pertussis toxin, indicating that extracellular sphingosine-1-phosphate via endogenously expressed G(i)-coupled receptors induces a stimulation of intracellular sphingosine-1-phosphate production. As sphingosine-1-phosphate- and sphingosylphosphorylcholine-induced increases in intracellular Ca2+ were blunted by sphingosine kinase inhibitors, this sphingosine-1-phosphate production appears to mediate Ca2+ signalling by extracellular sphingosine-1-phosphate and sphingosylphosphorylcholine in HEK-293 cells.

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors.

Sphingosine‐1‐phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G‐protein‐coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca2+ in intact cells independently of S1P‐GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca2+ concentration in HEK‐293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca2+ transients induced by photolysis of caged S1P were caused by Ca2+ mobilization from thapsigargin‐sensitive stores. These results provide direct evidence for a true intracellular action of S1P.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well‐studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X‐linked IAP (XIAP) and cellular IAP1 (c‐IAP1) directly bind to Rac1 in a nucleotide‐independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c‐IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1‐dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

Sphingosine-1-phosphate reduces rat renal and mesenteric blood flow in vivo in a pertussis toxin-sensitive manner

Sphingolipids such as sphingosine‐1‐phosphate (SPP) and sphingosylphosphorylcholine constrict isolated rat intrarenal and mesenteric microvessels in vitro. The present study investigates their effects on the cardiovascular system in vivo in anaesthetized rats. The animals were given intravenous or intrarenal arterial bolus injections of sphingolipids (0.1–100 μg kg−1) with subsequent measurements of mean arterial pressure, heart rate and renal and mesenteric blood flows (RBF, MBF) using a pressure transducer and electromagnetic flow probes, respectively. Intravenous injection of SPP rapidly (within 30 s), transiently and dose‐dependently reduced RBF (maximally −4.0±0.3 ml min−1) and MBF (maximally −1.4±0.2 ml min−1), without affecting mean arterial pressure or heart rate. Other sphingolipids had no significant effect. Intrarenal arterial SPP administration caused greater blood flow reductions (maximally −6.4±0.3 ml min−1) than systemic administration. Upon intrarenal administration, sphingosylphos‐ phorylcholine also lowered RBF (maximally −2.8±0.6 ml min−1), while the other sphingolipids remained without effect. Pretreatment with pertussis toxin (PTX, 10 μg kg−1) 3 days before the acute experiment abolished the SPP‐induced reductions of RBF and MBF. These data demonstrate, that SPP is a potent vasoconstrictor in vivo, particularly in the renal vasculature, while the other structurally related sphingolipids had little if any effects. The PTX‐sensitivity strongly suggests that the effects of SPP on renal and mesenteric blood flow are mediated by receptors coupled to Gi‐type G‐proteins.

Role of sphingosine kinase in Ca2+ signalling by epidermal growth factor receptor

Contribution of sphingosine kinase (SPK)‐catalyzed production of sphingosine‐1‐phosphate (SPP), in comparison to phospholipase C (PLC), to Ca2+ signalling by epidermal growth factor (EGF) was studied in two HEK‐293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca2+ by intracellular SPP. In HEK3 cells, EGF increased [Ca2+]i and stimulated both, SPK and PLC. [Ca2+]i increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca2+]i or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca2+ signalling by EGF in HEK‐293 cells.