Enkhzol Malchinkhuu

High-Density Lipoprotein Stimulates Endothelial Cell Migration and Survival Through Sphingosine 1-Phosphate and Its Receptors

Objective—Plasma high-density lipoprotein (HDL) level is inversely correlated with the risk of atherosclerosis. However, the cellular mechanism by which HDL exerts antiatherogenic actions is not well understood. In this study, we focus on the lipid components of HDL as mediators of the lipoprotein-induced antiatherogenic actions. Methods and Results—HDL and sphingosine 1-phosphate (S1P) stimulated the migration and survival of human umbilical vein endothelial cells. These responses to HDL and S1P were almost completely inhibited by pertussis toxin and other specific inhibitors for intracellular signaling pathways, although the inhibition profiles of migration and survival were different. The HDL-stimulated migration and survival of the cells were markedly inhibited by antisense oligonucleotides against the S1P receptors EDG-1/S1P1 and EDG-3/S1P3. Cell migration was sensitive to both receptors, but cell survival was exclusively sensitive to S1P1. The S1P-rich fraction and chromatographically purified S1P from HDL stimulated cell migration, but the rest of the fraction did not, as was the case of the cell survival. Conclusions—HDL-induced endothelial cell migration and survival may be mediated by the lipoprotein component S1P and the lipid receptors S1P1 and S1P3.

Critical role of ABCA1 transporter in sphingosine 1‐phosphate release from astrocytes

Sphingosine 1‐phosphate (S1P) is accumulated in lipoproteins, especially high‐density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL‐like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP‐binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP‐induced S1P release in association with the blockage of HDL‐like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up‐regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.

Role of p38 mitogen-activated kinase and c-Jun terminal kinase in migration response to lysophosphatidic acid and sphingosine-1-phosphate in glioma cells

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and S1P on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P1 to inhibition through S1P2. LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA1 receptor antagonists including Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways including Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signal-regulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominant-negative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/Rac1/JNK pathways are equally important for LPA1 receptor-mediated migration.

Identification of autotaxin as a neurite retraction-inducing factor of PC12 cells in cerebrospinal fluid and its possible sources

Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long‐acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long‐acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso‐PLD). Although the lyso‐PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso‐PLD activity and an ∼120‐kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso‐PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso‐PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.

LPA1 receptors mediate stimulation, whereas LPA2 receptors mediate inhibition, of migration of pancreatic cancer cells in response to lysophosphatidic acid and malignant ascites

Malignant ascites from pancreatic cancer patients has been reported to stimulate migration of pancreatic cancer cells through lysophosphatidic acid (LPA) and LPA(1) receptors. Indeed, ascites- and LPA-induced migration was inhibited by Ki16425, an LPA(1) and LPA(3) antagonist, in Panc-1 cells. Unexpectedly, however, in the presence of Ki16425, ascites and LPA inhibited cell migration in response to epidermal growth factor (EGF). The inhibitory migratory response to ascites and LPA was also observed in the cells treated with pertussis toxin (PTX), a G(i) protein inhibitor, and attenuated by a small interfering RNA (siRNA) specific to the LPA(2) receptor. The inhibitory LPA action was reversed by the regulators of G-protein signaling domain of p115RhoGEF, dominant-negative RhoA or C3 toxin. Indeed, LPA activated RhoA, which was attenuated by the siRNA against the LPA(2) receptor. Moreover, LP-105, an LPA(2) agonist, also inhibited EGF-induced migration in the PTX-treated cells. A similar inhibitory migration response through LPA(2) receptors was also observed in YAPC-PD, BxPC-3, CFPAC-1 and PK-1 pancreatic cancer cell lines. LPA also inhibited the invasion of Panc-1 cells in the PTX-treated cells in the in vitro Matrigel invasion assay. We conclude that LPA(2) receptors are coupled to the G(12/13) protein/Rho-signaling pathway, leading to the inhibition of EGF-induced migration and invasion of pancreatic cancer cells.

Assessment of the role of sphingosine 1-phosphate and its receptors in high-density lipoprotein-induced stimulation of astroglial cell function.

It has been suggested that lipoproteins in the central nervous system are involved in the regulation of several neural functions independent of cholesterol metabolism as well as those related to lipid metabolism. We recently demonstrated that lipoproteins are carriers for sphingosine 1-phosphate (S1P). This raised the possibility that S1P mediates the neural cell functions induced by lipoproteins. In the current study, we examined the effects of plasma high-density lipoprotein (HDL) on astroglial cell functions, focusing especially on the role of the lipoprotein-associated S1P. In rat type I astrocytes or C6 glioma cells, similar to S1P, HDL stimulated DNA synthesis and mRNA expression of fibroblast growth factor-2, a potent neurotrophic factor, which was associated with the activation of extracellular signal-regulated kinase (ERK) in a pertussis toxin-sensitive manner. The data from fractionation studies of HDL indicated that S1P may be a major component for the activation of ERK. In C6 glioma cells, HDL also induced phospholipase C-dependent intracellular Ca(2+) mobilization. Desensitization of the C6 glioma cells with S1P abolished these HDL-induced actions. Furthermore, overexpression of S1P receptors in C6 glioma cells led to a significant enhancement of HDL-induced ERK activation and Ca(2+) mobilization. Thus, at least some HDL-induced actions may be mediated by cell-surface S1P receptors in astroglial cells. These results imply that S1P might partially mediate lipoprotein-induced cholesterol metabolism-independent neural cell functions in the central nervous system.

Role of Rap1B and tumor suppressor PTEN in the negative regulation of lysophosphatidic acid--induced migration by isoproterenol in glioma cells.

The clarification of mechanisms that negatively regulate the invasive behavior of human glioma cells is of great importance in order to find new methods of treatment. In this study, we have focused on the negative regulation of lysophosphatidic acid (LPA)-induced migration in glioma cells. Using small interference RNA and dominant-negative gene strategies in addition to pharmacological tools, we found that isoproterenol (ISO) and sphingosine-1-phosphate (S1P) negatively but differently regulate the LPA-induced migration. ISO-induced suppression of the migration of glioma cells occurs via beta(2)-adrenergic receptor/cAMP/Epac/Rap1B/inhibition of Rac, whereas S1P has been shown to suppress the migration of the cells through S1P(2) receptor/Rho-mediated down-regulation of Rac1. The expression of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is required for the inhibitory ISO-induced and Rap1B-mediated actions on the migration, Rac1 activation, and Akt activation in response to LPA. Thus, the PTEN-mediated down-regulation of phosphatidylinositol 3-kinase activity may be involved in the regulation of Rap1B-dependent inhibition of Rac1 activity. These findings suggest that there are at least two distinct inhibitory pathways, which are mediated by the S1P(2) receptor and beta(2)-adrenergic receptor, to control the migratory, hence invasive, behavior of glioma cells.

Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1β-induced attenuation of Rho signaling pathways in rat astrocytes

J. Neurochem. (2011) 117, 164–174.

Signaling pathways involved in DNA synthesis and migration in response to lysophosphatidic acid and low-density lipoprotein in coronary artery smooth muscle cells

Low-density lipoprotein (LDL) and lysophosphatidic acid (LPA), one of the lipid components of lipoprotein, induced the DNA synthesis of coronary artery smooth muscle cells (CASMCs). The LDL- and LPA-induced DNA synthesis was markedly inhibited by the LPA receptor antagonist Ki16425, pertussis toxin, small interfering RNAs targeted for LPA1 receptors, and a potent calcineurin inhibitor cyclosporine A. It has been reported that LDL and LPA induced a migration response in a manner sensitive to Ki16425, pertussis toxin, and a LPA1 receptor-specific small interfering RNA. However, cyclosporine A was ineffective in inhibiting the migration response. Instead, an epidermal growth factor (EGF) receptor tyrosine kinase inhibitor markedly suppressed the migration response to LDL and LPA without having any significant effect on DNA synthesis. Thus, the LDL-induced stimulation of DNA synthesis and migration in CASMCs is mediated by its component LPA through LPA1 receptors and G(i/o)-proteins. Ca2+/calcineurin pathways and transactivation of EGF receptors mediate LPA1-receptor-induced DNA synthesis and migration, respectively.