Exogenous dihydrosphingosine 1 phosphate mediates collagen synthesis in cardiac fibroblasts through JAK/STAT signalling and regulation of TIMP1

Cardiac fibrosis and myocyte hypertrophy are hallmarks of the cardiac remodelling process in cardiomyopathies such as heart failure (HF). Dyslipidemia or dysregulation of lipids contribute to HF. The dysregulation of high density lipoproteins (HDL) could lead to altered levels of other lipid metabolites that are bound to it such as sphingosine-1- phosphate (S1P). Recently, it has been shown that S1P and its analogue dihydrosphingosine-1-phosphate (dhS1P) are bound to HDL in plasma. The effects of dhS1P on cardiac cells have been obscure. In this study, we show that extracellular dhS1P is able to increase collagen synthesis in neonatal rat cardiac fibroblasts (NCFs) and cause hypertrophy of neonatal cardiac myocytes (NCMs). The janus kinase/signal transducer and activator (JAK/STAT) signalling pathway was involved in the increased collagen synthesis by dhS1P, through sustained increase of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Extracellular dhS1P increased phosphorylation levels of STAT1 and STAT3 proteins, also caused an early increase in gene expression of transforming growth factor-β (TGFβ), and sustained increase in TIMP1. Inhibition of JAKs led to inhibition of TIMP1 and TGFβ gene and protein expression. We also show that dhS1P is able to cause NCM hypertrophy through S1P-receptor-1 (S1PR1) signalling which is opposite to that of its analogue, S1P. Taken together, our results show that dhS1P increases collagen synthesis in cardiac fibroblasts causing fibrosis through dhS1P-JAK/STAT-TIMP1 signalling.     

Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.     

Translational aspects of sphingosine 1-phosphate biology

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has both physiological and pathophysiological roles. It regulates cellular processes such as proliferation, migration, survival and differentiation and affects all organ systems. S1P not only activates S1P-specific receptors to initiate cellular signalling pathways but also directly regulates specific intracellular target proteins. The therapeutic opportunities surrounding S1P signalling are numerous and exemplified by the recent approval of FTY720 (a sphingosine analogue, Gilenya?) for the treatment of relapsing multiple sclerosis. A major focus of research is to develop small-molecule antagonists/agonists/inhibitors that are specific to the different S1P receptor subtypes and the enzymes that regulate S1P levels. This review describes fundamental aspects of S1P biology with an emphasis on the translational potential of intervention therapeutics.     

Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S(P)- and R(P)-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R(P)-enantiomer of the sarin analogue (k(cat)=2600 s(-1)) was the preferred substrate for the wild-type PTE relative to the corresponding S(P)-enantiomer (k(cat)=290 s(-1)). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k(cat) values for the R(P)- and S(P)-enantiomers were 410 and 4200 s(-1), respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R(P)R(C)-stereoisomer of the soman analogue was the preferred substrate for PTE. The k(cat) values for the soman analogues were measured as follows: R(P)R(C,) 48 s(-1); R(P)S(C), 4.8 s(-1); S(P)R(C), 0.3 s(-1), and S(P)S(C), 0.04 s(-1). With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k(cat) values for the soman analogues were found to be as follows: R(P)R(C,) 0.3 s(-1); R(P)S(C), 0.3 s(-1); S(P)R(C), 11s(-1), and S(P)S(C), 2.1 s(-1). Prior investigations have demonstrated that the S(P)-enantiomers of sarin and soman are significantly more toxic than the R(P)-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.     

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.     

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.     

Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

Fingolimod inhibits PDGF-B-induced migration of vascular smooth muscle cell by down-regulating the S1PR1/S1PR3 pathway

Platelet Derived Growth Factor (PDGF) and sphingosine-1-phosphate (S1P) pathways play a key role in mural cell recruitment during tumor growth and angiogenesis. Fingolimod, a S1P analogue, has been shown to exert antitumor and antiangiogenic properties. However, molecular targets and modes of action of fingolimod remain unclear. In this study, we confirmed the antagonizing action of S1P and PDGF-B on rat vascular smooth muscle cell (VSMCs) growth and migration. We then compared siRNA and/or fingolimod (100 nM) treatments on PDGFR-β, S1PR1 S1PR2 and S1PR3 expression. Fingolimod induced a 50% reduction in S1PR3 protein expression which was cumulative with that obtained with anti-S1PR3 siRNA. We found that siRNA-induced inhibition of both PDGFR-β and S1PR3 was the most effective means to block VSMC migration induced by PDGF-B. Finally, we observed that fingolimod treatment associated with anti-S1PR1 siRNA principally inhibited VSMC growth while in combination with anti-S1PR3 siRNA it strongly inhibited VSMC migration. These results suggest that for rat VSMCs, the PDGFR-S1PR1 pathway is predominantly dedicated to cell growth while PDGFR-S1PR3 stimulates cell migration. As an S1P analogue, fingolimod is considered a potent activator of S1PR1 and S1PR3. However, its action on the PDGFR-S1PR platform appears to be dependent on S1PR1 and S1PR3 specific downregulation. Considering that the S1P pathway has already been shown to exert various crosstalks with tyrosine kinase pathways, it seems of great interest to evaluate fingolimod potential in combination with the numerous tyrosine kinase inhibitors used in oncology.     

To stay or to leave: Stem cells and progenitor cells navigating the S1P gradient

Most hematopoietic stem progenitor cells (HSPCs) reside in bone marrow (BM), but a small amount of HSPCs have been found to circulate between BM and tissues through blood and lymph. Several lines of evidence suggest that sphingosine-1-phosphate (S1P) gradient triggers HSPC egression to blood circulation after mobilization from BM stem cell niches. Stem cells also visit certain tissues. After a temporary 36 h short stay in local tissues, HSPCs go to lymph in response to S1P gradient between lymph and tissue and eventually enter the blood circulation. S1P also has a role in the guidance of the primitive HSPCs homing to BM in vivo, as S1P analogue FTY720 treatment can improve HSPC BM homing and engraftment. In stress conditions, various stem cells or progenitor cells can be attracted to local injured tissues and participate in local tissue cell differentiation and tissue rebuilding through modulation the expression level of S1P1, S1P2 or S1P3 receptors. Hence, S1P is important for stem cells circulation in blood system to accomplish its role in body surveillance and injury recovery.     

Induction of antiproliferative connective tissue growth factor expression in Wilms' tumor cells by sphingosine-1-phosphate receptor 2

Connective tissue growth factor (CTGF), a member of the CCN family of secreted matricellular proteins, regulates fibrosis, angiogenesis, cell proliferation, apoptosis, tumor growth, and metastasis. However, the role of CTGF and its regulation mechanism in Wilms' tumor remains largely unknown. We found that the bioactive lipid sphingosine-1-phosphate (S1P) induced CTGF expression in a concentration- and time-dependent manner in a Wilms' tumor cell line (WiT49), whereas FTY720-phosphate, an S1P analogue that binds all S1P receptors except S1P2, did not. Further, the specific S1P2 antagonist JTE-013 completely inhibited S1P-induced CTGF expression, whereas the S1P1 antagonist VPC44116 did not, indicating that this effect was mediated by S1P2. This was confirmed by adenoviral transduction of S1P2 in WiT49 cells, which showed that overexpression of S1P2 increased the expression of CTGF. Induction of CTGF by S1P was sensitive to ROCK inhibitor Y-27632 and c-Jun NH2-terminal kinase inhibitor SP600125, suggesting the requirement of RhoA/ROCK and c-Jun NH2-terminal kinase pathways for S1P-induced CTGF expression. Interestingly, the expression levels of CTGF were decreased in 8 of 10 Wilms' tumor tissues compared with matched normal tissues by quantitative real-time PCR and Western blot analysis. In vitro, human recombinant CTGF significantly inhibited the proliferation of WiT49 cells. In addition, overexpression of CTGF resulted in significant inhibition of WiT49 cell growth. Taken together, these data suggest that CTGF protein induced by S1P2 might act as a growth inhibitor in Wilms' tumor.     

Metabolism and biological functions of two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate

Sphingolipids are major lipid constituents of the eukaryotic plasma membrane. Without certain sphingolipids, cells and/or embryos cannot survive, indicating that sphingolipids possess important physiological functions that are not substituted for by other lipids. One such role may be signaling. Recent studies have revealed that some sphingolipid metabolites, such as long-chain bases (LCBs; sphingosine (Sph) in mammals), long-chain base 1-phosphates (LCBPs; sphingosine 1-phosphate (S1P) in mammals), ceramide (Cer), and ceramide 1-phosphate (C1P), act as signaling molecules. The addition of phosphate groups to LCB/Sph and Cer generates LCBP/S1P and C1P, respectively. These phospholipids exhibit completely different functions than those of their precursors. In this review, we describe recent advances in understanding the functions of LCBP/S1P and C1P in mammals and in the yeast Saccharomyces cerevisiae. Since LCB/Sph, LCBP/S1P, Cer, and C1P are mutually convertible, regulation of not only the total amount of the each lipid but also of the overall balance in cellular levels is important. Therefore, we describe in detail their metabolic pathways, as well as the genes involved in each reaction.     

Inhibition of Ca(2+) signalling by the sphingosine 1-phosphate receptor S1P(1)

The lysophospholipid, sphingosine 1-phosphate (S1P), regulates a multitude of cellular functions by activating specific G protein-coupled receptors (GPCRs) (S1P(1-5), plus three newly identified S1P receptors). The G(i)-coupled S1P(1) receptor inhibits adenylyl cyclase, stimulates mitogen-activated protein kinases (MAP kinases) and cell migration, and is required for blood vessel maturation. Here, we report that S1P(1) inhibits Ca(2+) signalling in a number of cell types. In HEK-293 cells, which endogenously express S1P(1-3), overexpression of S1P(1) reduced intracellular free Ca(2+) concentration ([Ca(2+)](i)) increases induced by various receptor agonists as well as thapsigargin. The inhibitory Ca(2+) signalling of S1P(1) was blocked by pertussis toxin (PTX) and the protein kinase C (PKC) inhibitor, G?6976, and imitated by phorbol ester and overexpression of classical PKC isoforms. Activation of S1P(1) stably expressed in RH7777 cells, which endogenously do not express S1P receptors, also inhibited Ca(2+) signalling, without mediating Ca(2+) mobilization on its own. It is concluded that the widely expressed S1P receptor S1P(1) inhibits Ca(2+) signalling, most likely via G(i) proteins and classical PKC isoforms. Co-expression of S1P(1) with S1P(3), but not S1P(2), reversed the inhibitory effect of S1P(1), furthermore suggesting a specific interplay of S1P receptor subtypes usually found within a single cell type.     

Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.     

Follicular fluid high-density lipoprotein-associated sphingosine 1-phosphate (S1P) promotes human granulosa lutein cell migration via S1P receptor type 3 and small G-protein RAC1

Coordinated migration and progesterone production by granulosa cells is critical to the development of the corpus luteum, but the underlying mechanisms remain obscure. Sphingosine 1-phosphate (S1P), which is associated with follicular fluid high-density lipoprotein (FF-HDL), was previously shown to regulate ovarian angiogenesis. We herein examined the effects of S1P and FF-HDL on the function of granulosa lutein cells. Both FF-HDL and S1P induced migration of primary human granulosa lutein cells (hGCs) and the granulosa lutein cell line HGL5. In addition, FF-HDL but not S1P promoted progesterone synthesis, and neither of the two compounds stimulated proliferation of granulosa lutein cells. Polymerase chain reaction and Western blot experiments demonstrated the expression of S1P receptor type 1 (S1PR1), S1PR2, S1PR3, and S1PR5 but not S1PR4 in hGCs and HGL5 cells. The FF-HDL- and S1P-induced granulosa lutein cell migration was emulated by FTY720, an agonist of S1PR1, S1PR3, S1PR4, and S1PR5, and by VPC24191, an agonist of S1PR1 and S1PR3, but not by SEW2871 and phytosphingosine 1-phosphate, agonists of S1PR1 and S1PR4, respectively. In addition, blockade of S1PR3 with CAY1044, suramine, or pertussis toxin inhibited hGC and HGL5 cell migration toward FF-HDL or S1P, while blockade of S1PR1 and S1PR2 with W146 and JTE013, respectively, had no effect. Both FF-HDL and S1P triggered activation of small G-protein RAC1 and actin polymerization in granulosa cells, and RAC1 inhibition with Clostridium difficile toxin B or NSC23766 abolished FF-HDL- and S1P-induced migration. The FF-HDL-associated S1P promotes granulosa lutein cell migration via S1PR3 and RAC1 activation. This may represent a novel mechanism contributing to the development of the corpus luteum.     

Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection

Sphingosine-1-phosphate (S1P) induces an initial Ca(2+)-dependent contraction followed by a sustained Ca(2+)-independent, RhoA-mediated contraction in rabbit gastric smooth muscle cells. The cells coexpress S1P(1) and S1P(2) receptors, but the signaling pathways initiated by each receptor type and the involvement of one or both receptors in contraction are not known. Lentiviral vectors encoding small interfering RNAs were transiently transfected into cultured smooth muscle cells to silence S1P(1) or S1P(2) receptors. Phospholipase C (PLC)-beta activity and Rho kinase activity were used as markers of pathways mediating initial and sustained contraction, respectively. Silencing of S1P(1) receptors abolished S1P-stimulated activation of Galpha(i3) and partially inhibited activation of Galpha(i1), whereas silencing of S1P(2) receptors abolished activation of Galpha(q), Galpha(13), and Galpha(i2) and partially inhibited activation of Galpha(i1). Silencing of S1P(2) but not S1P(1) receptors suppressed S1P-stimulated PLC-beta and Rho kinase activities, implying that both signaling pathways were mediated by S1P(2) receptors. The results obtained by receptor silencing were corroborated by receptor inactivation. The selective S1P(1) receptor agonist SEW2871 did not stimulate PLC-beta or Rho kinase activity or induce initial and sustained contraction; when this agonist was used to protect S1P(1) receptors so as to enable chemical inactivation of S1P(2) receptors, S1P did not elicit contraction, confirming that initial and sustained contraction was mediated by S1P(2) receptors. Thus S1P(1) and S1P(2) receptors are coupled to distinct complements of G proteins. Only S1P(2) receptors activate PLC-beta and Rho kinase and mediate initial and sustained contraction.     

Role of charge and hydrophobic effects in reactions of peptide substrates and inhibitors with thrombin

A substrate and inhibitor analysis of the thrombin interaction with synthetic peptide substrates and inhibitors of differing hydrophobicity and volume of the side amino acid residue, localized in the sub-centers thrombin S2 and S3 were carried out. The kinetic parameters of individual stages of the enzymatic reaction process (K(S), k2, k3) were estimated. It is shown that the efficiency of acylation and deacylation stages of the enzymatic reaction decreases with increasing hydrophobicity of the substituent in P2 as well as P3, at the same time the affinity of selected peptides toward enzyme is steadily increasing. With the aim to evaluate the hydrophobicity of compounds a LogP value was calculated and was made an attempt to compare them with the correspondent Ki values. Comparative kinetic analysis of Z-Arg-OMe and its uncharged analogue Z-Cit-OMe has shown the absence of uncharged analog hydrolysis, however, the mentioned citrulline derivate inhibits the hydrolysis of the charged analogue. These findings confirm the important role of hydrophobic moiety in the structure of thrombin inhibitors in preferential binding mode and inhibition of thrombin active side.