Proliferation of hepatic myofibroblasts (hMF) is central for the development of fibrosis during liver injury, and factors that may limit their growth are potential antifibrotic agents. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with growth-regulating properties, either via Edg receptors or through intracellular actions. In this study, we examined the effects of S1P on the proliferation of human hMF. Human hMF expressed mRNAs for the S1P receptors Edg1, Edg3, and Edg5. These receptors were functional at nanomolar concentrations and coupled to pertussis toxin-sensitive and -insensitive G proteins, as demonstrated in guanosine 5-3-O-(thio)triphosphate binding assays. S1P potently inhibited hMF growth (IC 50 ‫؍‬ 1 M), in a pertussis toxininsensitive manner. Analysis of the mechanisms involved in growth inhibition revealed that S1P rapidly increased prostaglandin E 2 production and in turn cAMP, two growth inhibitory messengers for hMF; C 2 -ceramide and sphingosine, which inhibited hMF proliferation, did not affect cAMP levels. Production of cAMP by S1P was abolished by NS-398, a selective inhibitor of COX-2. Also, S1P potently induced COX-2 protein expression. Blocking COX-2 by NS-398 blunted the antiproliferative effect of S1P. We conclude that S1P inhibits proliferation of hMF, probably via an intracellular mechanism, through early COX-2-dependent release of prostaglandin E 2 and cAMP, and delayed COX-2 induction. Our results shed light on a novel role for S1P as a growth inhibitory mediator and point out its potential involvement in the negative regulation of liver fibrogenesis.

Hepatic myofibroblasts (hMFs) are central in the development of liver fibrosis during chronic liver diseases, and their removal by apoptosis contributes to the resolution of liver fibrosis. We previously identified Edg receptors for sphingosine 1-phosphate (S1P) in human hMFs. Here, we investigated the effects of S1P on hMF apoptosis. S1P reduced viability of serum-deprived hMFs by an apoptotic process that was unrelated to the conversion of S1P into sphingosine and ceramide. The apoptotic effects of S1P were receptor-independent because dihydro-S1P, an Edg agonist, had no effect. S1P also stimulated a receptor-dependent survival pathway, revealed by enhanced activation of caspase-3 by S1P in the presence of pertussis toxin. Cell survival relied on two pertussis toxin-sensitive events, activation of ERK and activation of phosphatidylinositol 3-kinase (PI3K)/ Akt by S1P. Both pathways were also activated by dihydro-S1P. Blunting either ERK or PI3K enhanced caspase-3 stimulation by S1P, and simultaneous inhibition of both pathways resulted in additive effects on caspase-3 activation. In conclusion, S1P induces apoptosis of human hMFs via a receptor-independent mechanism and stimulates a survival pathway following activation of Edg receptors. The survival pathway arises from the sequential activation of G i /G o proteins and independent stimulations of ERK and PI3K/Akt. Therefore, blocking Edg receptors may sensitize hepatic myofibroblasts to apoptosis by S1P.Liver fibrosis is the common response to chronic liver injury and is characterized by increased deposition and altered composition of extracellular matrix. This fibrogenic process is consecutive to intense proliferation and accumulation of myofibroblasts that synthesize fibrosis components and proinflammatory cytokines (1). Apoptosis of hepatic myofibroblasts is emerging as a key event in the regression of liver fibrogenesis. Indeed, it has been shown in experimental models of liver fibrosis that withdrawal of the offending agent is associated with apoptosis of hepatic myofibroblasts, followed by activation of fibrolysis mechanisms and regression of fibrosis (2). Therefore, identification of factors that govern death and survival of liver fibrogenic cells is of crucial interest for the design of antifibrotic therapies. Several apoptotic factors for these cells have recently been identified, such as soluble Fas (CD95/ APO-1) ligand, nerve growth factor, benzodiazepines, gliotoxin, and 15-deoxy-⌬ 12,14 -prostaglandin J2 (3-8). Recent lines of evidence suggest that sphingolipids, in addition to being structural constituents of cell membranes, play a key role as signaling molecules. In this respect, metabolites of sphingolipids, including ceramide, sphingosine, and sphingosine 1-phosphate (S1P), 1 have emerged as a new class of lipid messengers that regulate cell proliferation, differentiation, and survival (9 -11). Ceramide and sphingosine are generated after receptor-coupled activation of sphingomyelinase and ceramidase, respectively, and have generally been lin...

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Hepatic myofibroblasts (hMFs) play a key role in the development of liver fibrosis associated with chronic liver diseases. Apoptosis of these cells is emerging as a key process in the resolution of liver fibrosis. Here, we examined the effects of cyclopentenone prostaglandins on apoptosis of human hMFs. Cyclopentenone prostaglandins of the J series markedly reduced hMF viability, with 15-deoxy-⌬ 12,14-prostaglandin J2 (15-d-PGJ 2) being the most potent. This effect was independent of peroxisome-proliferator-activated receptors (PPARs), because PPAR␥ and PPAR␣ agonists did not affect hMF cell viability, and PPAR␥, the nuclear receptor for 15-d-PGJ 2 , was not expressed in hMFs. Moreover, 15-d-PGJ 2 did not act via a cell surface G protein-coupled receptor, as shown in guanosine-5-O-(3-thiotriphosphate) binding assays. Cell death resulted from an apoptotic process, because 15-d-PGJ 2-treated hMFs exhibited condensed nuclei, fragmented DNA, and elevated caspase-3 activity. Moreover, the caspase inhibitor Z-Val-Ala-Asp (OCH 3)-fluoromethyl ketone blocked the cytotoxic effect of 15-d-PGJ 2. The apoptotic effects of 15-d-PGJ2 were reproduced by H 2 O 2 and blocked by the antioxidants N-acetylcysteine (NAC), N-(2-mercapto-propionyl)glycine (NMPG) and pyrrolidine dithiocarbamate (PDTC). Accordingly, 15-d-PGJ 2 generated rapid production of reactive oxygen species in hMFs, via a NAC/ NMPG/PDTC-sensitive pathway. In conclusion, 15-d-PGJ 2 induces apoptosis of human hMFs via a novel mechanism involving oxidative stress and unrelated to activation of its nuclear receptor PPAR␥. These data underline the antifibrogenic potential of 15-d-PGJ 2 .