Docosatetraenoyl LPA is elevated in exhaled breath condensate in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective medical therapies. Recent research has focused on identifying the biological processes essential to the development and progression of fibrosis, and on the mediators driving these processes. Lysophosphatidic acid (LPA), a biologically active lysophospholipid, is one such mediator. LPA has been found to be elevated in bronchoalveolar lavage (BAL) fluid of IPF patients, and through interaction with its cell surface receptors, it has been shown to drive multiple biological processes implicated in the development of IPF. Accordingly, the first clinical trial of an LPA receptor antagonist in IPF has recently been initiated. In addition to being a therapeutic target, LPA also has potential to be a biomarker for IPF. There is increasing interest in exhaled breath condensate (EBC) analysis as a non-invasive method for biomarker detection in lung diseases, but to what extent LPA is present in EBC is not known. METHODS: In this study, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess for the presence of LPA in the EBC and plasma from 11 IPF subjects and 11 controls. RESULTS: A total of 9 different LPA species were detectable in EBC. Of these, docosatetraenoyl (22:4) LPA was significantly elevated in the EBC of IPF subjects when compared to controls (9.18 pM vs. 0.34 pM; p = 0.001). A total of 13 different LPA species were detectable in the plasma, but in contrast to the EBC, there were no statistically significant differences in plasma LPA species between IPF subjects and controls. CONCLUSIONS: These results demonstrate that multiple LPA species are detectable in EBC, and that 22:4 LPA levels are elevated in the EBC of IPF patients. Further research is needed to determine the significance of this elevation of 22:4 LPA in IPF EBC, as well as its potential to serve as a biomarker for disease severity and/or progression.

Targeting sphingosine kinase 1 attenuates bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor ß (TGF-ß) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-ß secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-ß dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.

Role of palmitate-induced sphingoid base-1-phosphate biosynthesis in INS-1 ß-cell survival.

Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic ß cells and what role they play in palmitate-induced ß cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 ß cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated ß-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of ß cells plays a protective role against palmitate-induced ceramide-dependent apoptotic ß cell death.

Protection of LPS-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression.

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Dynamin 2 and c-Abl are novel regulators of hyperoxia-mediated NADPH oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.

Phospholipase D-mediated activation of IQGAP1 through Rac1 regulates hyperoxia-induced p47phox translocation and reactive oxygen species generation in lung endothelial cells.

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47(phox) to cell periphery, and ROS production. Exposure of [(32)P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O(2) and 5% CO(2)) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [(32)P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O(2)(.)/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [(32)P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47(phox) redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47(phox) translocation and ROS formation in human lung endothelial cells.

Regulation of COX-2 expression and IL-6 release by particulate matter in airway epithelial cells.

Particulate matter (PM) in ambient air is a risk factor for human respiratory and cardiovascular diseases. The delivery of PM to airway epithelial cells has been linked to release of proinflammatory cytokines; however, the mechanisms of PM-induced inflammatory responses are not well-characterized. This study demonstrates that PM induces cyclooxygenase (COX)-2 expression and IL-6 release through both a reactive oxygen species (ROS)-dependent NF-kappaB pathway and an ROS-independent C/EBPbeta pathway in human bronchial epithelial cells (HBEpCs) in culture. Treatment of HBEpCs with Baltimore PM induced ROS production, COX-2 expression, and IL-6 release. Pretreatment with N-acetylcysteine (NAC) or EUK-134, in a dose-dependent manner, attenuated PM-induced ROS production, COX-2 expression, and IL-6 release. The PM-induced ROS was significantly of mitochondrial origin, as evidenced by increased oxidation of the mitochondrially targeted hydroethidine to hydroxyethidium by reaction with superoxide. Exposure of HBEpCs to PM stimulated phosphorylation of NF-kappaB and C/EBPbeta, while the NF-kappaB inhibitor, Bay11-7082, or C/EBPbeta siRNA attenuated PM-induced COX-2 expression and IL-6 release. Furthermore, NAC or EUK-134 attenuated PM-induced activation of NF-kappaB; however, NAC or EUK-134 had no effect on phosphorylation of C/EBPbeta. In addition, inhibition of COX-2 partly attenuated PM-induced Prostaglandin E2 and IL-6 release.

Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells.

In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.

Lysophosphatidic acid-induced transactivation of epidermal growth factor receptor regulates cyclo-oxygenase-2 expression and prostaglandin E(2) release via C/EBPbeta in human bronchial epithelial cells.

We have demonstrated that LPA (lysophosphatidic acid)-induced IL (interleukin)-8 secretion was partly mediated via transactivation of EGFR [EGF (epidermal growth factor) receptor] in HBEpCs (human bronchial epithelial primary cells). The present study provides evidence that LPA-induced transactivation of EGFR regulates COX (cyclo-oxygenase)-2 expression and PGE(2) [PG (prostaglandin) E(2)] release through the transcriptional factor, C/EBPbeta (CCAAT/enhancer-binding protein beta), in HBEpCs. Treatment with LPA (1 microM) stimulated COX-2 mRNA and protein expression and PGE(2) release via G(alphai)-coupled LPARs (LPA receptors). Pretreatment with inhibitors of NF-kappaB (nuclear factor-kappaB), JNK (Jun N-terminal kinase), or down-regulation of c-Jun or C/EBPbeta with specific siRNA (small interference RNA) attenuated LPA-induced COX-2 expression. Downregulation of EGFR by siRNA or pretreatment with the EGFR tyrosine kinase inhibitor, AG1478, partly attenuated LPA-induced COX-2 expression and phosphorylation of C/EBPbeta; however, neither of these factors had an effect on the NF-kappaB and JNK pathways. Furthermore, LPA-induced EGFR transactivation, phosphorylation of C/EBPbeta and COX-2 expression were attenuated by overexpression of a catalytically inactive mutant of PLD2 [PLD (phospholipase D) 2], PLD2-K758R, or by addition of myristoylated PKCzeta [PKC (protein kinase C) zeta] peptide pseudosubstrate. Overexpression of the PLD2-K758R mutant also attenuated LPA-induced phosphorylation and activation of PKCzeta. These results demonstrate that LPA induces COX-2 expression and PGE(2) production through EGFR transactivation-independent activation of transcriptional factors NF-kappaB and c-Jun, and EGFR transactivation-dependent activation of C/EBPbeta in HBEpCs. Since COX-2 and PGE(2) have been shown to be anti-inflammatory in airway inflammation, the present data suggest a modulating and protective role of LPA in regulating innate immunity and remodelling of the airways.

De novo biosynthesis of dihydrosphingosine-1-phosphate by sphingosine kinase 1 in mammalian cells.

Sphingosine kinase 1 (SK1) is one of the two known kinases, which generates sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator involved in cell survival, proliferation, and cell-cell interactions. Activation of SK1 and intracellular generation of S1P were suggested to be part of the growth and survival factor-induced signaling, and overexpression of SK1 provoked cell tumorigenic transformation. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant upregulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by deviating metabolic flow of newly formed sphingoid bases from ceramide formation toward the synthesis of DHS1P. On the contrary, S1P biosynthesis was not directly linked to the de novo sphingoid bases transformations and was dependent on catabolic generation of sphingosine from complex sphingolipids. As a result of SK1 overexpression, migration and Ca2+-response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, was strongly impaired. In contrast, selective increase in intracellular content of DHS1P or S1P through the uptake and phosphorylation of corresponding sphingoid bases had no effect on S1P-induced signaling or facilitation of wound healing. Furthermore, infection of human bronchial epithelial cells (HBEpC) with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-alpha enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can control survival/death (DHS1P-S1P/ceramides) balance by targeting sphingolipid de novo biosynthesis and selectively generating DHS1P at a metabolic step preceding ceramide formation.

Marine alkaloid polycarpine and its synthetic derivative dimethylpolycarpine induce apoptosis in JB6 cells through p53- and caspase 3-dependent pathways.

PURPOSE: Polycarpine from ascidian Polycarpa aurata was previously found to be active against different human tumor cells. In this study, we investigated the antitumor mechanisms of polycarpine and its synthetic derivative, desmethoxyethoxy-polycarpine (dimethylpolycarpine), through the induction of apoptosis. This new knowledge regarding the proapoptotic action of polycarpine and dimethylpolycarpine should lead to a better understanding of their effects and development of a new class of anticancer drugs. METHODS: Apoptosis was clearly observed by flow cytometry and Western blotting using an antibody against cleaved caspase-3 as an apoptotic marker. RESULTS: Polycarpines differentially activated p38 kinase, JNKs, and ERKs in JB6 Cl 41 cells. The polycarpines-induced apoptosis was decreased in cells expressing a dominant-negative mutant of JNK. Both compounds stimulated p53-dependent transcriptional activity and phosphorylation. Induction of p53-phosphorylation at serine 15 was suppressed in JNKI and JNK2 knockout cells. Furthermore, polycarpines were unable to induce apoptosis in p53-deficient MEFs in contrast to a strong induction of apoptosis in wild type MEFs, suggesting that p53 is involved in apoptosis induced by polycarpines. The p53 phosphorylation in turn was mediated by activated JNKs. CONCLUSIONS: These results indicate that all three MAPK signaling pathways are involved in the response of JB6 cells to treatment with polycarpines. Evidence also supports a proapoptotic role of the JNKs signaling pathway in vivo and clearly indicates that JNKs are required for phosphorylation of c-Jun, activation of p53, and subsequent apoptosis induced by polycarpines.

A new cytotoxic fatty acid (5Z,9Z)-22-methyl-5,9-tetracosadienoic acid and the sterols from the far eastern sponge Geodinella robusta.

A new fatty acid, (5Z,9Z)-22-methyl-5,9-tetracosadienoic acid (1a), and a rare fatty acid, (5Z,9Z)-23-methyl-5,9-tetracosadienoic acid (2a), the predominant constituents of the free fatty acid fraction from the lipids of the sponge Geodinella robusta, were isolated and partly separated by reversed phase high-performance liquid chromatography, followed by multifold crystallization from MeOH to give 1a and 2a in 70% and 60% purity, respectively. These fatty acids were identified as (5Z,9Z)-22- and (5Z,9Z)-23-methyl-5,9-tetracosadienoic acids by nuclear magnetic resonance techniques, including distortionless enhancement by polarization transfer, heteronuclear multiple quantum connectivity, and correlation spectroscopy experiments, as well as from mass-spectrometric data for their methyl esters, the methyl esters of their perhydro derivatives, and their pyrrolidides. Mixtures of 1a and 2a showed cytotoxic activity against mouse Ehrlich carcinoma cells and a hemolytic effect on mouse erythrocytes. The sterol fraction from the same sponge was analyzed by gas-liquid chromatography-mass spectrometry, and 24-methylenecholesterol was identified as a main constituent of this fraction. The implications of the co-occurrence of membranolytic long-chain fatty acids and 24-methylenecholesterol as a main membrane sterol are discussed in terms of the phenomenon of biochemical coordination.