Contrary effects of sphingosine-1-phosphate on expression of α-smooth muscle actin in transforming growth factor ß1-stimulated lung fibroblasts.

Transforming growth factor-ß1 (TGFß1) plays a pivotal role in fibrosis in various organs including the lung. Following pulmonary injury, TGFß1 stimulates conversion of fibroblasts to myofibroblasts that are mainly characterized by up-regulation of α-smooth muscle actin (αSMA) expression, and the resulting excess production of extracellular matrix proteins causes fibrosis with loss of alveolar function. The present study was undertaken to define the role of the sphingosine-1-phosphate (S1P) pathway in TGFß1-induced expression of αSMA in human fetal lung fibroblasts, HFL1 cells. Analysis of mRNA revealed the existence of S1P(1), S1P(2), and S1P(3) receptor mRNAs. Treatment with TGFß1 increased sphingosine kinase (SphK) activity and S1P(3) receptor mRNA at 24h after stimulation, and pharmacological data showed the involvement of sphingomyelinase, SphK, and S1P(3) receptor in the TGFß1-induced up-regulation of αSMA with and without serum. Treatment with pertussis toxin and S1P(1) receptor antagonist W146 enhanced αSMA expression by TGFß1/serum, and S1P decreased and increased αSMA levels with and without serum, respectively. TGFß1 increased cyclooxygenase-2 expression in a manner dependent on serum and the sphingomyelinase/SphK pathway, and the response was decreased by pertussis toxin. Prostaglandin E(2), formed by TGFß1/serum stimulation, decreased the TGFß1-induced expression of αSMA via EP prostanoid receptor. These data suggest that S1P formed by TGFß1 stimulation has diverse effects on the expression of αSMA, inhibition via the S1P(1) receptor-mediated and serum-dependent expression of cyclooxygenase-2 and the resulting formation of prostaglandin E(2), and stimulation via the S1P(3) receptor in a serum-independent manner.

Inhibitory effects of clinical reagents having anti-oxidative activity on transforming growth factor-ß1-induced expression of α-smooth muscle actin in human fetal lung fibroblasts.

Excessive production of transforming growth factor-ß1 (TGFß1) plays an important role in lung fibrosis, in which the differentiation of fibroblasts into myofibroblasts is a key process. Increased formation of reactive oxygen species (ROS) induced by TGFß1 is a common pathological feature of fibrosis. In the present study, probucol and lovastatin, which are therapeutics used for hyperlipidemia and proposed to act as anti-oxidants, were examined in terms of their effect on TGFß1-induced formation of ROS and expression of α-smooth muscle actin (αSMA), a myofibroblast marker, in human fetal lung fibroblasts (HFL1 cells). The effects of anti-oxidative enzymes and reagents including N-acetyl-L-cysteine, α-tocopherol, and lecithinized-superoxide dismutase (SOD) on TGFß1-induced expression of αSMA were also examined. Treatment with probucol (30 µM) and lovastatin (1 µM and 3 µM), in addition to lecithinized-SOD, significantly inhibited the TGFß1-induced formation of ROS and αSMA. Other anti-oxidants including N-acetyl-L-cysteine had marginal effects on αSMA expression under the conditions. Probucol and lovastatin, established therapeutics, may be worth trying in patients with both lung fibrosis and hypercholesterolemia.

Newly synthetic ceramide-1-phosphate analogs; their uptake, intracellular localization, and roles as an inhibitor of cytosolic phospholipase A(2)α and inducer of cell toxicity.

Ceramide-1-phosphate (C1P) regulates cellular functions including arachidonic acid (AA) metabolism and modulates cell fate. The mechanism by which C1P is taken up is unclear, and the development of lipophilic analogs may be useful for regulating C1P's actions. We synthesized new mono- and di-methyl-ester (MM and DM, respectively) analogs of C1P with N-acyl chains of different lengths, and examined their effects on AA release and cell toxicity. Short-N-acyl-DM-C1P analogs including C5- and C6-DM-C1P, but not long-N-acyl-DM-C1P analogs, inhibited the release of AA mediated by α type cytosolic phospholipase A(2) (cPLA(2)α) in Chinese hamster ovary (CHO) cells and the enzymatic activity. Short-N-acyl-DM-C1P analogs including C6-DM-C1P caused morphological changes with cell toxicity 24h after the treatment in three cells lines (CHO, L929, and RLC-18 cells), although the role of AA in the toxicity was not clear. Neither long-N-acyl-DM-C1P analogs nor MM-C1P analogs including C6-MM-C1P affected cPLA(2)α activity and cell toxicity. Similar to C6-ceramide having a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) group on a C6-N-acyl chain (NBD-C6-ceramide), NBD-C6-DM-C1P and C6-DM-C1P-NBD (with a C6-N-acyl chain and an NBD-labeled C14-alkyl chain) were accumulated in the Golgi complex, although less C6-DM-C1P-NBD than NBD-C6-DM-C1P was taken up. NBD-C6-ceramide was converted to various metabolites including NBD-C6-sphingomyelin, but both NBD-C6-DM-C1P and C6-DM-C1P-NBD were stable in cells within 2h. The short-N-acyl-DM-C1P analogs acted directly as an inhibitor of cPLA(2)α and an inducer of cell toxicity, and may be useful for the regulation of ceramide/C1P-regulated responses.

Effects of synthetic sphingosine-1-phosphate analogs on cytosolic phospholipase A2alpha-independent release of arachidonic acid and cell toxicity in L929 fibrosarcoma cells: the structure-activity relationship.

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3'-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type alpha cytosolic phospholipase A(2) (cPLA(2)alpha)] and L929-cPLAalpha-siRNA cells (L929 cells treated with small interference RNA for cPLA(2)alpha). Treatment with pharmacological inhibitors of secretory and Ca(2+)-independent PLA(2)s decreased the DMB-mC11S-induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3'-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA(2) activity and its toxicity in cells. [Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP].

Cytotoxicity induced by inhibition of thioredoxin reductases via multiple signaling pathways: role of cytosolic phospholipase A(2)alpha-dependent and -independent release of arachidonic acid.

The thioredoxin (Trx) system, comprising Trx, the selenoprotein thioredoxin reductase (TrxR), and NADPH, functions as an antioxidant system. Trx has various biological activities including growth control and anti-apoptotic properties, and the Trx system offers a target for the development of drugs to treat and/or prevent cancer. We evaluated the role of TrxR inhibition in the release of arachidonic acid (AA), cell toxicity, and intracellular signaling pathways in L929 mouse fibrosarcoma cells. Treatment with 1-chloro-2,4-dinitrobenzene (DNCB, an inhibitor of TrxR) under conditions involving limited inhibition of TrxR activity in cells, released AA before causing cytotoxicity. Treatment with an inhibitor of p38 kinase, a downstream enzyme of the apoptosis signal-regulating kinase 1 pathway, and pyrrophenone (an inhibitor of alpha-type cytosolic phospholipase A(2), cPLA(2)alpha) partially but significantly decreased the DNCB-induced release of AA and cell death. The responses were much weaker in cPLA(2)alpha knockdown L929 cells. Exogenously added AA showed cytotoxicity. DNCB increased intracellular reactive oxygen species (ROS) levels, and butylated hydroxyanisole (an antioxidant) reduced DNCB-induced ROS formation and cell toxicity but not the phosphorylation of p38 kinase and release of AA. Auranofin, another inhibitor of TrxR having a different formula, released AA resulting in toxicity in L929 cells. DNCB caused the release of AA and cytotoxicity in A549 human lung carcinoma cells, and caused p38 kinase-dependent toxicity in PC12 rat pheochromocytoma cells. Our data suggest that a dysfunctional Trx system triggers multiple signaling pathways, and that the AA released by cPLA(2)alpha-dependent and -independent pathways is important to cytotoxicity. J. Cell. Physiol. 219: 606-616, 2009. (c) 2009 Wiley-Liss, Inc.