Glycolipid transfer protein knockout disrupts vesicle trafficking to the plasma membrane.

The glycolipid transfer protein (GLTP) has been linked to many cellular processes aside from its best-known in vitro function as a lipid transport protein. It has been proposed to act as a sensor and regulator of glycosphingolipid homeostasis in cells. Furthermore, through its previously determined interaction with the endoplasmic reticulum membrane protein VAP-A (vesicle-associated membrane protein-associated protein A), GLTP may also be involved in facilitating vesicular transport in cells. In this study, we characterized the phenotype of CRISPR/Cas9-mediated GLTP KO HeLa cells. We showed that motility, three-dimensional growth, and cellular metabolism were all altered by GLTP knockout. Expression of a GLTP mutant incapable of binding VAP disrupted cell spheroid formation, indicating that the GLTP-VAP interaction is linked to cellular adhesion, cohesion, and three-dimensional growth. Most notably, we found evidence that GLTP, through its interaction with VAP-A, affects vesicular trafficking, marking the first cellular process discovered to be directly impacted by a change in GLTP expression.

Lysosome Associated Protein Transmembrane 4B-24 Is the Predominant Protein Isoform in Human Tissues and Undergoes Rapid, Nutrient-Regulated Turnover.

Studies of lysosome associated protein transmembrane 4B (LAPTM4B) have mainly focused on the 35-kDa isoform and its association with poor prognosis in cancers. Here, by employing a novel monoclonal antibody, the authors found that the 24-kDa LAPTM4B isoform predominated in most, both healthy and malignant, human cells and tissues studied. LAPTM4B-24 lacks the extreme N-terminus and, contrary to LAPTM4B-35, failed to promote cell migration. The endogenous LAPTM4B-24 protein was subject to rapid turnover with a t1/2 of approximately 1 hour. The protein was degraded by both lysosomal and proteasomal pathways, and its levels were increased by the availability of nutrients and lysosomal ceramide. These findings underscore the pathophysiological relevance of the LAPTM4B-24 isoform and identify it as a dynamically regulated effector in lysosomal nutrient signaling.

LAPTM4B facilitates late endosomal ceramide export to control cell death pathways.

Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.

Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5) disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs) harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X), were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.

A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²âº microdomain in the caveolar compartment.

Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1-Aequorin fusion protein (Cav1-Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca²âº]cav). In HeLa cells, Cav1-Aeq reported different [Ca²âº] as compared to the plasma membrane [Ca²âº] in general (reported by SNAP25-Aeq) or as compared to the cytosolic [Ca²âº] (reported by cyt-Aeq). The Ca²âº signals detected by Cav1-Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-ß-cyclodextrin, suggesting that the caveolae are specific targets for Ca²âº signaling. HeLa cells overexpressing SK1 showed increased [Ca²âº]cav during histamine-induced Ca²âº mobilization in the absence of extracellular Ca²âº as well as during receptor-operated Ca²âº entry (ROCE). The SK1-induced increase in [Ca²âº]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca²âº]cav during ROCE. S1P treatment stimulated the [Ca²âº]cav upon ROCE. The Ca²âº responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca²âº signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

NDRG1 functions in LDL receptor trafficking by regulating endosomal recycling and degradation.

N-myc downstream-regulated gene 1 (NDRG1) mutations cause Charcot-Marie-Tooth disease type 4D (CMT4D). However, the cellular function of NDRG1 and how it causes CMT4D are poorly understood. We report that NDRG1 silencing in epithelial cells results in decreased uptake of low-density lipoprotein (LDL) due to reduced LDL receptor (LDLR) abundance at the plasma membrane. This is accompanied by the accumulation of LDLR in enlarged EEA1-positive endosomes that contain numerous intraluminal vesicles and sequester ceramide. Concomitantly, LDLR ubiquitylation is increased but its degradation is reduced and ESCRT (endosomal sorting complex required for transport) proteins are downregulated. Co-depletion of IDOL (inducible degrader of the LDLR), which ubiquitylates the LDLR and promotes its degradation, rescues plasma membrane LDLR levels and LDL uptake. In murine oligodendrocytes, Ndrg1 silencing not only results in reduced LDL uptake but also in downregulation of the oligodendrocyte differentiation factor Olig2. Both phenotypes are rescued by co-silencing of Idol, suggesting that ligand uptake through LDLR family members controls oligodendrocyte differentiation. These findings identify NDRG1 as a novel regulator of multivesicular body formation and endosomal LDLR trafficking. The deficiency of functional NDRG1 in CMT4D might impair lipid processing and differentiation of myelinating cells.

Tracking sphingosine metabolism and transport in sphingolipidoses: NPC1 deficiency as a test case.

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3)H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3)H]-sphingomyelin-derived [(3)H]-ceramide and depletion of [(3)H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3)H]-sphingosine-derived [(3)H]-ceramide and attenuation of [(3)H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3)H]-sphingosine derived from [(3)H]-sphingomyelin/LDL or [(3)H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3)H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.

LAPTM4B counteracts ferroptosis via suppressing the ubiquitin-proteasome degradation of SLC7A11 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics.

miR-137-LAPTM4B regulates cytoskeleton organization and cancer metastasis via the RhoA-LIMK-Cofilin pathway in osteosarcoma.

Osteosarcoma (OS) is a rare malignant bone tumor but is one leading cause of cancer mortality in childhood and adolescence. Cancer metastasis accounts for the primary reason for treatment failure in OS patients. The dynamic organization of the cytoskeleton is fundamental for cell motility, migration, and cancer metastasis. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is an oncogene participating in various biological progress central to cancer biogenesis. However, the potential roles of LAPTM4B in OS and the related mechanisms remain unknown. Here, we established the elevated LAPTM4B expression in OS, and it is essential in regulating stress fiber organization through RhoA-LIMK-cofilin signaling pathway. In terms of mechanism, our data revealed that LAPTM4B promotes RhoA protein stability by suppressing the ubiquitin-mediated proteasome degradation pathway. Moreover, our data show that miR-137, rather than gene copy number and methylation status, contributes to the upregulation of LAPTM4B in OS. We report that miR-137 is capable of regulating stress fiber arrangement, OS cell migration, and metastasis via targeting LAPTM4B. Combining results from cells, patients' tissue samples, the animal model, and cancer databases, this study further suggests that the miR-137-LAPTM4B axis represents a clinically relevant pathway in OS progression and a viable target for novel therapeutics.

Cln5-deficiency in mice leads to microglial activation, defective myelination and changes in lipid metabolism.

CLN5 disease, late infantile variant phenotype neuronal ceroid lipofuscinosis, is a severe neurodegenerative disease caused by mutations in the CLN5 gene, which encodes a lysosomal protein of unknown function. Cln5-deficiency in mice leads to loss of thalamocortical neurons, and glial activation, but the underlying mechanisms are poorly understood. We have now studied the gene expression of Cln5 in the mouse brain and show that it increases gradually with age and differs between neurons and glia, with the highest expression in microglia. In Cln5(-/-) mice, we documented early and significant microglial activation that was already evident at 3 months of age. Loss of Cln5 also leads to defective myelination in vitro and in the developing mouse brain. This was accompanied by early alterations in serum lipid profiles, dysfunctional cellular metabolism and lipid transport in Cln5(-/-) mice. Taken together, these data provide significant new information about events associated with Cln5-deficiency, revealing altered myelination and disturbances in lipid metabolism, together with an early neuroimmune response.

FTY720 stimulates 27-hydroxycholesterol production and confers atheroprotective effects in human primary macrophages.

RATIONALE: The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known. OBJECTIVE: We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages. METHODS AND RESULTS: FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann-Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor-induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not. CONCLUSIONS: We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.

LAPTM4B controls the sphingolipid and ether lipid signature of small extracellular vesicles.

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.

An autocrine sphingosine-1-phosphate signaling loop enhances NF-kappaB-activation and survival.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a multitude of cellular functions, including cell proliferation, survival, migration and angiogenesis. S1P mediates its effects either by signaling through G protein-coupled receptors (GPCRs) or through an intracellular mode of action. In this study, we have investigated the mechanism behind S1P-induced survival signalling. RESULTS: We found that S1P protected cells from FasL-induced cell death in an NF-kappaB dependent manner. NF-kappaB was activated by extracellular S1P via S1P2 receptors and Gi protein signaling. Our study also demonstrates that extracellular S1P stimulates cells to rapidly produce and secrete additional S1P, which can further amplify the NF-kappaB activation. CONCLUSIONS: We propose a self-amplifying loop of autocrine S1P with capacity to enhance cell survival. The mechanism provides increased understanding of the multifaceted roles of S1P in regulating cell fate during normal development and carcinogenesis.

Overexpression of TRPC3 reduces the content of intracellular calcium stores in HEK-293 cells.

The mammalian canonical transient receptor channels (TRPCs) are considered to be candidates for store-operated calcium channels (SOCCs). Many studies have addressed how TRPC3 channels are affected by depletion of intracellular calcium stores. Conflicting results have been shown for TRPC3 regarding its function, and this has been linked to its level of expression in various systems. In the present study, we have investigated how overexpression of TRPC3 interferes with the regulation of intracellular calcium stores. We demonstrate that overexpression of TRPC3 reduces the mobilization of calcium in response to stimulation of the cells with thapsigargin (TG) or the G-protein coupled receptor agonist sphingosine-1-phosphate (S1P). Our results indicate that this is the result of the expression of TRPC3 channels in the endoplasmic reticulum (ER), thus depleting ER calcium stores. OAG evoked calcium entry in cells overexpressing TRPC3, indicating that functional TRPC3 channels were also expressed in the plasma membrane. Taken together, our results show that overexpression of the putative SOCC, TRPC3, actually reduces the calcium content of intracellular stores, but does not enhance agonist-evoked or store-dependent calcium entry. Our results may, in part, explain the conflicting results obtained in previous studies on the actions of TRPC3 channels.

A Ceramide-Regulated Element in the Late Endosomal Protein LAPTM4B Controls Amino Acid Transporter Interaction.

Membrane proteins are functionally regulated by the composition of the surrounding lipid bilayer. The late endosomal compartment is a central site for the generation of ceramide, a bioactive sphingolipid, which regulates responses to cell stress. The molecular interactions between ceramide and late endosomal transmembrane proteins are unknown. Here, we uncover in atomistic detail the ceramide interaction of Lysosome Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent cell death and autophagy, and its functional relevance in lysosomal nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends on a sphingolipid interaction motif and an adjacent aspartate residue in the protein's third transmembrane (TM3) helix. The interaction motif provides the preferred contact points for ceramide while the neighboring membrane-embedded acidic residue confers flexibility that is subject to ceramide-induced conformational changes, reducing TM3 bending. This facilitates the interaction between LAPTM4B and the amino acid transporter heavy chain 4F2hc, thereby controlling mTORC signaling. These findings provide mechanistic insights into how transmembrane proteins sense and respond to ceramide.

Antiproliferative effect of sphingosylphosphorylcholine in thyroid FRO cancer cells mediated by cell cycle arrest in the G2/M phase.

Among the group of bioactive sphingolipids, sphingosylphosphorylcholine (SPC) has been known to induce both antiproliferative and proliferative effects depending on cell type. In the present investigation we show that SPC (1-10 microM) reduced the proliferation of FRO cells (an anaplastic thyroid carcinoma cell line) in a concentration dependent manner. The effect was pertussis toxin insensitive, and independent of phospholipase C, protein kinase C, p38 kinase, or jun kinase. In addition to inhibiting the migration of FRO cells, application of SPC induced a rapid (<10 min) rounding of the cells, which was dependent on extracellular sodium. However, DAPI staining and caspase-3 analysis could not reveal any apoptotic effects of SPC. Furthermore, when cells treated with SPC for 24h were washed and replated, they continued to grow, albeit somewhat slower than control cells. Flow cytometry analysis revealed a significant increase in the population of cells in the G2-M phase, and a reduction in S phase. SPC reduced the phosphorylation of Akt with about 50% and evoked a substantial decrease in the amount of phosphorylated mitogen-activated protein (MAP) kinase. In cells treated with the PI3 kinase inhibitor wortmannin, both migration and proliferation were inhibited, as well as the amount of phosphorylated MAP kinase. Treatment of the cells with either SPC or wortmannin increased the levels of p21, but decreased that of cyclin B1 and Cdc2. Taken together, SPC is an effective suppressor of thyroid cancer cell proliferation and migration, and this effect is, in part, mediated by inhibition of both the PI3K-Akt and the MAP kinase signalling pathways.

Sphingosine kinase regulates voltage operated calcium channels in GH4C1 rat pituitary cells.

We have previously shown that sphingosine inhibits depolarisation-induced calcium influx through voltage-operated calcium channels (VOCCs) in GH(4)C(1) cells, whereas sphingosine-1-phosphate (S1P) does not. In the present study we investigated whether sphingosine kinase modulates VOCC activity in GH(4)C(1) cells by removing inhibitory sphingosine. Sphingosine and the structurally similar sphingosine kinase inhibitor dimethylsphingosine (DMS) both rapidly attenuated the calcium influx evoked by depolarisation. The inhibitory effect declined over time to a greater extent in cells treated with sphingosine than in cells treated with DMS, indicating that sphingosine is being metabolised more rapidly. When the specific sphingosine kinase inhibitor 2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKi) was added to the cells after depolarisation there was likewise a reduction of the calcium response. This inhibitory effect was slow and reached a plateau about 3 min after application. In contrast, the sphingosine-mediated inhibition was immediate, suggesting that the SKi-induced inhibition was due to build-up of cellular sphingosine. In experiments on cells overexpressing sphingosine kinase, the inhibitory effect of sphingosine was reversed faster than in control cells. The effect was not due to the produced S1P, since S1P did not have any effect on VOCCs even at concentrations as high as 50 microM. In patch-clamp experiments the calcium entry through VOCCs was attenuated in GH(4)C(1) cells overexpressing a kinase-dead sphingosine kinase, compared with cells overexpressing the wild type sphingosine kinase. In addition, in cells treated with SKi the calcium entry through VOCCs was attenuated compared with control cells. Our results provide compelling evidence that sphingosine kinase regulates the function of voltage-operated calcium channels in GH(4)C(1) cells, not through its catalytic product, but by removal of the substrate sphingosine.

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C.

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.

Enhancement of intracellular sphingosine-1-phosphate production by inositol 1,4,5-trisphosphate-evoked calcium mobilisation in HEK-293 cells: endogenous sphingosine-1-phosphate as a modulator of the calcium response.

Sphingosine-1-phosphate (S1P) regulates many cellular functions, such as migration, differentiation and growth. The effects of S1P are thought to be primarily mediated by G-protein coupled receptors, but an intracellular function as a calcium releasing second messenger has also been proposed. Here we show that in HEK-293 cells, exogenous S1P mobilises sequestered calcium by a mechanism primarily dependent on the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) pathway, and secondarily on the subsequent synthesis of intracellular S1P. Stimulating HEK-293 cells exogenously with S1P increased the production of both inositol phosphates and intracellular S1P. The calcium response was inhibited in cells treated with 2-APB, caffeine or U73122, showing that the PLC/IP3 pathway for calcium release is activated in response to exogenous S1P. The calcium response was partially inhibited in cells treated with the sphingosine kinase inhibitor DMS and in cells expressing a catalytically inactive sphingosine kinase, showing that endogenously produced S1P is also involved. Importantly, 2-APB and U73122 inhibited the S1P-evoked production of intracellular S1P. S1P is therefore not likely a major calcium releasing second messenger in HEK-293 cells, but rather a secondary regulator of calcium mobilisation.