Resveratrol inhibits cell growth by inducing cell cycle arrest in activated hepatic stellate cells.

Resveratrol (RSV) exerts anti-proliferative and pro-apoptotic actions in different cell lines. Hepatic stellate cells (HSCs) are major fibrogenic cell types that contribute to collagen accumulation during chronic liver disease. In the present study, the inhibitory effects of RSV on cell proliferation, cell cycle, and apoptosis were evaluated in the mouse hepatic stellate cell line GRX. Cells treated with 1 nM-1 muM of RSV demonstrated a decrease in cell growth of about 35% after 5 days. GRX cells, treated with RSV (100 nM or 1 muM), were analyzed by flow cytometry; RSV induced an increase in the number of GRX cells in the S- and sub-G1 phases. The increase in sub-G1 phase cells and the nuclear condensation and fragmentation shown by DAPI staining identified a possible pro-apoptotic effect of RSV on GRX cells. Furthermore, the RSV anti-proliferative effects could be explained by an S-phase accumulation caused by a decrease in the progression through the cell cycle or an inhibition of S or G2 phase transition. It is notable that these RSV actions are mediated at nanomolar levels, compatible with the concentrations of free RSV in biological fluids after ingestion of polyphenol-rich foods, suggesting a possible effect of these foods as an adjuvant treatment in chronic liver diseases.

Activity and expression of ecto-5'-nucleotidase/CD73 are increased during phenotype conversion of a hepatic stellate cell line.

Hepatic stellate cells (HSC) play a crucial role in the development of liver fibrosis and are important targets in liver disease therapy. Adenosine acts as an extracellular signaling molecule in various tissues and in liver this nucleoside exerts protective effects. Ecto-5'-nucleotidase/CD73 is a marker for the plasma membrane and is considered to be a key enzyme in the generation of adenosine in the extracellular medium, by transforming AMP into adenosine. In addition, adenosine production from AMP is also catalyzed by alkaline phosphatase. We compared the extracellular metabolism of AMP and transcriptional levels of the ecto-5'-nucleotidase/CD73 and tissue non-specific alkaline phosphatase (TNALP) in activated and quiescent HSC of the mouse hepatic stellate cell line GRX. This cell line expresses a myofibroblast phenotype in basal medium and both retinol and indomethacin treatment induced a phenotypic change of GRX cells to quiescent HSC. Ecto-5'-nucleotidase activity and its mRNA expression were found to be higher in quiescent HSC than in activated HSC. During phenotype conversion, mediated by retinol, the AMP decay was accelerated with adenosine accumulation in extracellular medium, likely due to the decrease in adenosine deaminase activity also observed in quiescent HSC. The treatment with retinol also involves transcriptional activation of TNALP. Taken together, these data suggest that ecto-5'-nucleotidase-dependent adenosine generation may play a role in the regulation of quiescent HSC functions.

Hepatic stellate cell line modulates lipogenic transcription factors.

BACKGROUND/AIMS: Pre-adipocyte differentiation into adipocyte is a terminal differentiation process triggered by a cascade of transcription factors. Conversely, hepatic stellate cells (HSC) can switch between lipid storing and the myofibroblast phenotype in association with liver fibrotic processes. Here, adipogenic/lipogenic-related transcription factors and downstream-regulated genes were evaluated in a murine HSC cell line. GRX-HSC cells are transitional myofibroblasts that differentiate into lipocytes following retinol or indomethacin treatment. METHODS/RESULTS: Specific mRNAs were quantified by a real-time polymerase chain reaction after 24 h or 7 days of cell culture with indomethacin or retinol. Proliferator-activated receptorgamma and Pex16 transcripts were increased either by retinol or indomethacin. Retinol induced a minor increase in C/enhancer binding proteinalpha transcripts, while only indomethacin increased adipsin transcripts. CONCLUSIONS: Our results showed that the myofibroblast to lipocyte phenotype switch follows partially different transcriptional pathways, according to the effector. Retinol induces lipid synthesis and storage without affecting characteristic adipocytic genes, while indomethacin treatment restores the lipocytic phenotype with increased adipisin expression.

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line.

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM beta-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 muM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-(14)C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain's composition.

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells.

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.

Effect of pentoxifylline on arachidonic acid metabolism, neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell.

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A2 (PLA2), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1-2.5 microM), it increased the quantity of stored lipids. Retinol and PTF (5 microM and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA2 activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE2, but not of PGF2. However, when in presence of retinol, we observed a slightly higher increase in PGE2 and PGF2a production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.

Changes of sphingolipid species in the phenotype conversion from myofibroblasts to lipocytes in hepatic stellate cells.

Sphingolipids play a relevant role in cell-cell interaction, communication, and migration. We studied the sphingolipid content in the murine hepatic stellate cell line GRX, which expresses the myofibroblast phenotype, and can be induced in vitro to display the fat-storing phenotype. Lipid modifications along this induction were investigated by labeling sphingolipids with [(14)C]galactose, [(14)C]serine, or [(14)C]choline, and determination of fatty acid composition of sphingomyelin. The total ganglioside content and the GM2 synthase activity were lower in myofibroblasts. Both phenotypes presented similar gangliosides of the a-pathway: GM2, GM1, and GD1a as well as their precursor GM3. Sphingomyelin and all the gangliosides were expressed as doublets; the upper/lower band ratio increased in lipocytes, containing more long-chain fatty acids in retinol-induced lipocytes as compared to the insulin/indomethacin induced ones. Time-course experiments indicated a transfer of metabolic precursors from phosphatidylcholine to sphingomyelin in the two phenotypes. Taken together, these results indicate that myofibroblast and lipocytes can use distinct ceramide pools for sphingolipid synthesis. Differential ganglioside expression and presence of the long-chain saturated fatty acids suggested that they may participate in formation of distinct membrane microdomains or rafts with specific functions on the two phenotypes of GRX-cells.