David L. Ebenezer

Reactive oxygen species at the crossroads of inflammasome and inflammation

Inflammasomes form a crucial part of the innate immune system. These are multi-protein oligomer platforms that are composed of intracellular sensors which are coupled with caspase and interleukin activating systems. Nod-like receptor protein (NLRP) 3, and 6 and NLRC4 and AIM2 are the prominent members of the inflammasome family. Inflammasome activation leads to pyroptosis, a process of programmed cell death distinct from apoptosis through activation of Caspase and further downstream targets such as IL-1β and IL-18 leading to activation of inflammatory cascade. Reactive oxygen species (ROS) serves as important inflammasome activating signals. ROS activates inflammasome through mitogen-activated protein kinases (MAPK) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Dysregulation of inflammasome plays a significant role in various pathological processes. Viral infections such as Dengue and Respiratory syncytial virus activate inflammasomes. Crystal compounds in silicosis and gout also activate ROS. In diabetes, inhibition of autophagy with resultant accumulation of dysfunctional mitochondria leads to enhanced ROS production activating inflammasomes. Activation of inflammasomes can be dampened by antioxidants such as SIRT-1. Inflammasome and related cascade could serve as future therapeutic targets for various pathological conditions.

Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy

Introduction Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis. Methods S1P lyase (S1PL) expression in peripheral blood mononuclear cells (PBMCs) was correlated with pulmonary functions and overall survival; used a murine model to check the role of S1PL on the fibrogenesis and a cell culture system to study the effect of S1PL expression on transforming growth factor (TGF)-β- and S1P-induced fibroblast differentiation. Results S1PL expression was upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. TGF-β increased the expression of S1PL in human lung fibroblasts via activation and binding of Smad3 transcription factor to Sgpl1 promoter. Overexpression of S1PL attenuated TGF-β-induced and S1P-induced differentiation of human lung fibroblasts through regulation of the expression of LC3 and beclin 1. Knockdown of S1PL (Sgpl1+/−) in mice augmented bleomycin-induced pulmonary fibrosis, and patients with IPF reduced Sgpl1 mRNA expression in PBMCs exhibited higher severity of fibrosis and lower survival rate. Conclusion These studies suggest that S1PL is a novel endogenous suppressor of pulmonary fibrosis in human IPF and animal models.

Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis.

Targeting sphingosine-1-phosphate signaling in lung diseases.

Sphingosine-1-phosphate (S1P), a simple, bioactive sphingolipid metabolite, plays a key role, both intracellularly and extracellularly, in various cellular processes such as proliferation, survival, migration, inflammation, angiogenesis, and endothelial barrier integrity. The cellular S1P level is low and is tightly regulated by its synthesis and degradation. Sphingosine Kinases (SphKs) 1 and 2, catalyze the ATP-dependent phosphorylation of sphingosine to S1P, while the degradation is mediated by the reversible dephosphorylation catalyzed by the S1P phosphatases and lipid phosphate phosphatases and the irreversible degradation to hexadecenal and ethanolamine phosphate by sphingosine-1-phosphate lyase (S1PL). As a ligand for specific G-protein-coupled receptors, S1P1-5, which are differentially expressed in different cell types, S1P generates downstream signals that play crucial role in developmental and disease related pathologies. In addition to acting extracellularly on receptors located on the plasma membrane, S1P can also act intracellularly, independently of S1P1-5, affecting calcium homeostasis and cell proliferation. The SphKs /S1P /S1PL metabolic pathway is implicated in numerous human pathologies including respiratory disorders, thereby raising the possibility that manipulating intracellular S1P levels could offer therapeutic potential in ameliorating lung diseases. This review focuses on the prospects of targeting S1P signaling and S1P metabolizing enzymes using small molecule inhibitors, receptor agonists, and antagonists in the treatment of lung diseases.

Role of sphingosine kinase 1 and S1P transporter Spns2 in HGF-mediated lamellipodia formation in lung endothelium

Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P1, in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P1.

Hyperoxia- induced p47phox activation and ROS generation is mediated through S1P transporter Spns2, and S1P/S1P1&2 signaling axis in lung endothelium.

Hyperoxia-induced lung injury adversely affects ICU patients and neonates on ventilator assisted breathing. The underlying culprit appears to be reactive oxygen species (ROS)-induced lung damage. The major contributor of hyperoxia-induced ROS is activation of the multiprotein enzyme complex NADPH oxidase. Sphingosine-1-phosphate (S1P) signaling is known to be involved in hyperoxia-mediated ROS generation; however, the mechanism(s) of S1P-induced NADPH oxidase activation is unclear. Here, we investigated various steps in the S1P signaling pathway mediating ROS production in response to hyperoxia in lung endothelium. Of the two closely related sphingosine kinases (SphKs)1 and 2, which synthesize S1P from sphingosine, only Sphk1(-/-) mice conferred protection against hyperoxia-induced lung injury. S1P is metabolized predominantly by S1P lyase and partial deletion of Sgpl1 (Sgpl1(+/-)) in mice accentuated lung injury. Hyperoxia stimulated S1P accumulation in human lung microvascular endothelial cells (HLMVECs), and downregulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1&2, but not S1P3, using specific siRNA attenuated hyperoxia-induced p47(phox) translocation to cell periphery and ROS generation in HLMVECs. These results suggest a role for Spns2 and S1P1&2 in hyperoxia-mediated ROS generation. In addition, p47(phox) (phox:phagocyte oxidase) activation and ROS generation was also reduced by PF543, a specific SphK1 inhibitor in HLMVECs. Our data indicate a novel role for Spns2 and S1P1&2 in the activation of p47(phox) and production of ROS involved in hyperoxia-mediated lung injury in neonatal and adult mice.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia

BackgroundSphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1−/−) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array.ResultsTwo-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1−/− mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1−/− neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45.ConclusionUsing SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

Nuclear lipid mediators: Role of nuclear sphingolipids and sphingosine-1-phosphate signaling in epigenetic regulation of inflammation and gene expression

Phospholipids, sphingolipids, and cholesterol are integral components of eukaryotic cell organelles, including the nucleus. Recent evidence shows characteristic features of nuclear lipid composition and signaling, which are distinct from that of the cytoplasm and plasma membrane. While the nuclear phosphoinositol lipid signaling in cell cycle regulation and differentiation has been well described, there is a paucity on the role of nuclear sphingolipids and sphingolipid signaling in different physiological and pathophysiological human conditions. In this prospective, we describe the role of sphingolipids and specifically focus on the sphingoid bases, such as sphingosine, ceramide, and sphingosine‐1‐phosphate (S1P) generation and catabolism in nuclear signaling and function. Particularly, S1P generated in the nucleus by phosphorylation of SPHK2 modulates HDAC activity either by direct binding or through activation of nuclear reactive oxygen species and regulates cell cycle and pro‐inflammatory gene expression. Potential implication of association of SPHK2 with the co‐repressor complexes and generation of S1P in the nucleus on chromatin remodeling under normal and pathological conditions is discussed. A better understanding of sphingolipid signaling in the nucleus will facilitate the design and development of new and novel therapeutic approaches to modulate expression of pro‐inflammatory and cell cycle dependent genes in human pathologies such as cancer, bacterial lung infection, neurodegeneration, and cystic fibrosis.

Sphingolipids in Ventilator Induced Lung Injury: Role of Sphingosine-1-Phosphate Lyase

Mechanical ventilation (MV) performed in respiratory failure patients to maintain lung function leads to ventilator-induced lung injury (VILI). This study investigates the role of sphingolipids and sphingolipid metabolizing enzymes in VILI using a rodent model of VILI and alveolar epithelial cells subjected to cyclic stretch (CS). MV (0 PEEP (Positive End Expiratory Pressure), 30 mL/kg, 4 h) in mice enhanced sphingosine-1-phosphate lyase (S1PL) expression, and ceramide levels, and decreased S1P levels in lung tissue, thereby leading to lung inflammation, injury and apoptosis. Accumulation of S1P in cells is a balance between its synthesis catalyzed by sphingosine kinase (SphK) 1 and 2 and catabolism mediated by S1P phosphatases and S1PL. Thus, the role of S1PL and SphK1 in VILI was investigated using Sgpl1+/− and Sphk1−/− mice. Partial genetic deletion of Sgpl1 protected mice against VILI, whereas deletion of SphK1 accentuated VILI in mice. Alveolar epithelial MLE-12 cells subjected to pathophysiological 18% cyclic stretch (CS) exhibited increased S1PL protein expression and dysregulation of sphingoid bases levels as compared to physiological 5% CS. Pre-treatment of MLE-12 cells with S1PL inhibitor, 4-deoxypyridoxine, attenuated 18% CS-induced barrier dysfunction, minimized cell apoptosis and cytokine secretion. These results suggest that inhibition of S1PL that increases S1P levels may offer protection against VILI.

ID: 111: THE S1P TRANSPORTER, SPNS2, MEDIATES HGF-INDUCED LAMELLIPODIA FORMATION AND MIGRATION OF HUMAN LUNG ENDOTHELIAL CELLS

Rationale We have demonstrated earlier that HGF-induced lamellipodia formation in human lung microvascular endothelial cells (HLMVECs) was through c-Met receptor tyrosine kinase and PI3 kinase/Akt signal transduction. Here, we show that HGF-mediated lamellipodia formation is dependent on intracellular S1P generation mediated by sphingosine kinase 1 (SphK1), the S1P transporter, Spns2 and S1P1 in HLMVECs. Methods HLMVECs were treated with HGF (20 ng/ml) for different time points. Lamellipodia were detected after HGF treatment by immunofluorescent staining of Spns2, cortactin and actin in lamellipodia, and lamellipodia were quantified by measuring cell periphery fluorescence intensity. Pearsons correlation coefficient was used to statistically quantify co-localization of proteins in lamellipodia. Endogenous SphK activity was blocked by SphK1 specific inhibitor PF-543, and expression of SphK1 in cells was down-regulated by siRNA. Cellular S1P levels were quantified by mass spectrometry. Results HGF stimulated phosphorylation of SphK1, and its localization to lamellipodia of HLMVECs. Down-regulation of SphK1, but not SphK2, with siRNA or inhibition of SphK1 with PF-543 (1–5 µM) attenuated HGF-induced lamellipodia formation in HLMVECs. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on PI3K/Akt and ERK1/2 signaling apthways. HGF increased S1P levels in HLMVECs, which was blocked by inhibition of SphK1 with PF-543. Further, HGF induced serine phosphorylation and translocation of Spns2, the S1P transporter, to lamellipodia, which was Akt dependent. The HGF-induced lamellipodia formation in HLMVECs was blocked by down-regulation of Spns2, suggesting extracellular action of S1P in lamellipodia formation. Down-regulation of S1P1, but not S1P2 or S1P3, with siRNA attenuated HGF-induced lamellipodia formation. Further, HGF stimulation enhanced association of Spns2 with S1P1 and blocking SphK1 activty with PF-543 attenuated the association between Spns2 and S1P1. Additionally, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Conclusion These results suggest that HGF/c-Met mediated lamellipodia formation and motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2 mediated transport of S1P to outside for signaling via S1P1 in HLMVECs. This work was supported by NIH/HLBI P01 HL98050 to VN.