Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

The Role of Sphingolipid Signaling in Oxidative Lung Injury and Pathogenesis of Bronchopulmonary Dysplasia.

Premature infants are born with developing lungs burdened by surfactant deficiency and a dearth of antioxidant defense systems. Survival rate of such infants has significantly improved due to advances in care involving mechanical ventilation and oxygen supplementation. However, a significant subset of such survivors develops the chronic lung disease, Bronchopulmonary dysplasia (BPD), characterized by enlarged, simplified alveoli and deformed airways. Among a host of factors contributing to the pathogenesis is oxidative damage induced by exposure of the developing lungs to hyperoxia. Recent data indicate that hyperoxia induces aberrant sphingolipid signaling, leading to mitochondrial dysfunction and abnormal reactive oxygen species (ROS) formation (ROS). The role of sphingolipids such as ceramides and sphingosine 1-phosphate (S1P), in the development of BPD emerged in the last decade. Both ceramide and S1P are elevated in tracheal aspirates of premature infants of <32 weeks gestational age developing BPD. This was faithfully reflected in the murine models of hyperoxia and BPD, where there is an increased expression of sphingolipid metabolites both in lung tissue and bronchoalveolar lavage. Treatment of neonatal pups with a sphingosine kinase1 specific inhibitor, PF543, resulted in protection against BPD as neonates, accompanied by improved lung function and reduced airway remodeling as adults. This was accompanied by reduced mitochondrial ROS formation. S1P receptor1 induced by hyperoxia also aggravates BPD, revealing another potential druggable target in this pathway for BPD. In this review we aim to provide a detailed description on the role played by sphingolipid signaling in hyperoxia induced lung injury and BPD.

Sphingosine kinase 1 regulates lysyl oxidase through STAT3 in hyperoxia-mediated neonatal lung injury.

INTRODUCTION: Neonatal lung injury as a consequence of hyperoxia (HO) therapy and ventilator care contribute to the development of bronchopulmonary dysplasia (BPD). Increased expression and activity of lysyl oxidase (LOX), a key enzyme that cross-links collagen, was associated with increased sphingosine kinase 1 (SPHK1) in human BPD. We, therefore, examined closely the link between LOX and SPHK1 in BPD. METHOD: The enzyme expression of SPHK1 and LOX were assessed in lung tissues of human BPD using immunohistochemistry and quantified (Halo). In vivo studies were based on Sphk1-/- and matched wild type (WT) neonatal mice exposed to HO while treated with PF543, an inhibitor of SPHK1. In vitro mechanistic studies used human lung microvascular endothelial cells (HLMVECs). RESULTS: Both SPHK1 and LOX expressions were increased in lungs of patients with BPD. Tracheal aspirates from patients with BPD had increased LOX, correlating with sphingosine-1-phosphate (S1P) levels. HO-induced increase of LOX in lungs were attenuated in both Sphk1-/- and PF543-treated WT mice, accompanied by reduced collagen staining (sirius red). PF543 reduced LOX activity in both bronchoalveolar lavage fluid and supernatant of HLMVECs following HO. In silico analysis revealed STAT3 as a potential transcriptional regulator of LOX. In HLMVECs, following HO, ChIP assay confirmed increased STAT3 binding to LOX promoter. SPHK1 inhibition reduced phosphorylation of STAT3. Antibody to S1P and siRNA against SPNS2, S1P receptor 1 (S1P1) and STAT3 reduced LOX expression. CONCLUSION: HO-induced SPHK1/S1P signalling axis plays a critical role in transcriptional regulation of LOX expression via SPNS2, S1P1 and STAT3 in lung endothelium.

Hyperoxia-induced S1P1 signaling reduced angiogenesis by suppression of TIE-2 leading to experimental bronchopulmonary dysplasia.

INTRODUCTION: We have earlier shown that hyperoxia (HO)-induced sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling contribute to bronchopulmonary dysplasia (BPD). S1P acts through G protein-coupled receptors, S1P1 through S1P5. Further, we noted that heterozygous deletion of S1pr1 ameliorated the HO-induced BPD in the murine model. The mechanism by which S1P1 signaling contributes to HO-induced BPD was explored. METHODS: S1pr1+/+ and S1pr1+/- mice pups were exposed to either room air (RA) or HO (75% oxygen) for 7 days from PN 1-7. Lung injury and alveolar simplification was evaluated. Lung protein expression was determined by Western blotting and immunohistochemistry (IHC). In vitro experiments were performed using human lung microvascular endothelial cells (HLMVECs) with S1P1 inhibitor, NIBR0213 to interrogate the S1P1 signaling pathway. RESULTS: HO increased the expression of S1pr1 gene as well as S1P1 protein in both neonatal lungs and HLMVECs. The S1pr1+/- neonatal mice showed significant protection against HO-induced BPD which was accompanied by reduced inflammation markers in the bronchoalveolar lavage fluid. HO-induced reduction in ANG-1, TIE-2, and VEGF was rescued in S1pr1+/- mouse, accompanied by an improvement in the number of arterioles in the lung. HLMVECs exposed to HO increased the expression of KLF-2 accompanied by reduced expression of TIE-2, which was reversed with S1P1 inhibition. CONCLUSION: HO induces S1P1 followed by reduced expression of angiogenic factors. Reduction of S1P1 signaling restores ANG-1/ TIE-2 signaling leading to improved angiogenesis and alveolarization thus protecting against HO-induced neonatal lung injury.

Neonatal therapy with PF543, a sphingosine kinase 1 inhibitor, ameliorates hyperoxia-induced airway remodeling in a murine model of bronchopulmonary dysplasia.

Hyperoxia (HO)-induced lung injury contributes to bronchopulmonary dysplasia (BPD) in preterm newborns. Intractable wheezing seen in BPD survivors is associated with airway remodeling (AWRM). Sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling promotes HO-mediated neonatal BPD; however, its role in the sequela of AWRM is not known. We noted an increased concentration of S1P in tracheal aspirates of neonatal infants with severe BPD, and earlier, demonstrated that Sphk1-/- mice showed protection against HO-induced BPD. The role of SPHK1/S1P in promoting AWRM following exposure of neonates to HO was investigated in a murine model. Therapy using PF543, the specific SPHK1 inhibitor, during neonatal HO reduced alveolar simplification followed by reduced AWRM in adult mice. This was associated with reduced airway hyperreactivity to intravenous methacholine. Neonatal HO exposure was associated with increased expression of SPHK1 in lung tissue of adult mice, which was reduced with PF543 therapy in the neonatal stage. This was accompanied by amelioration of HO-induced reduction of E-cadherin in airway epithelium. This may be suggestive of arrested partial epithelial mesenchymal transition (EMT) induced by HO. In vitro studies using human primary airway epithelial cells (HAEpCs) showed that SPHK1 inhibition or deletion restored HO-induced reduction in E-cadherin and reduced formation of mitochondrial reactive oxygen species (mtROS). Blocking mtROS with MitoTempo attenuated HO-induced partial EMT of HAEpCs. These results collectively support a therapeutic role for PF543 in preventing HO-induced BPD in neonates and the long-term sequela of AWRM, thus conferring a long-term protection resulting in improved lung development and function.

Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts.

The sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-ß stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-ß-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-ß- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-ß-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-ß- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-ß-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

Advancements in understanding the role of lysophospholipids and their receptors in lung disorders including bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a devastating chronic neonatal lung disease leading to serious adverse consequences. Nearly 15 million babies are born preterm accounting for >1 in 10 births globally. The aetiology of BPD is multifactorial and the survivors suffer lifelong respiratory morbidity. Lysophospholipids (LPL), which include sphingosine-1-phosphate (S1P), and lysophosphatidic acid (LPA) are both naturally occurring bioactive lipids involved in a variety of physiological and pathological processes such as cell survival, death, proliferation, migration, immune responses and vascular development. Altered LPL levels have been observed in a number of lung diseases including BPD, which underscores the importance of these signalling lipids under normal and pathophysiological situations. Due to the paucity of information related to LPLs in BPD, most of the ideas related to BPD and LPL are speculative. This article is intended to promote discussion and generate hypotheses, in addition to the limited review of information related to BPD already established in the literature.

Pseudomonas aeruginosa stimulates nuclear sphingosine-1-phosphate generation and epigenetic regulation of lung inflammatory injury.

INTRODUCTION: Dysregulated sphingolipid metabolism has been implicated in the pathogenesis of various pulmonary disorders. Nuclear sphingosine-1-phosphate (S1P) has been shown to regulate histone acetylation, and therefore could mediate pro-inflammatory genes expression. METHODS: Profile of sphingolipid species in bronchoalveolar lavage fluids and lung tissue of mice challenged with Pseudomonas aeruginosa (PA) was investigated. The role of nuclear sphingosine kinase (SPHK)2 and S1P in lung inflammatory injury by PA using genetically engineered mice was determined. RESULTS: Genetic deletion of Sphk2, but not Sphk1, in mice conferred protection from PA-mediated lung inflammation. PA infection stimulated phosphorylation of SPHK2 and its localisation in epithelial cell nucleus, which was mediated by protein kinase C (PKC) δ. Inhibition of PKC δ or SPHK2 activity reduced PA-mediated acetylation of histone H3 and H4, which was necessary for the secretion of pro-inflammatory cytokines, interleukin-6 and tumour necrosis factor-α. The clinical significance of the findings is supported by enhanced nuclear localisation of p-SPHK2 in the epithelium of lung specimens from patients with cystic fibrosis (CF). CONCLUSIONS: Our studies define a critical role for nuclear SPHK2/S1P signalling in epigenetic regulation of bacterial-mediated inflammatory lung injury. Targeting SPHK2 may represent a potential strategy to reduce lung inflammatory pulmonary disorders such as pneumonia and CF.

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.

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

BACKGROUND: Sphingosine- 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. RESULTS: Two-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. CONCLUSION: Using 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.