HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 41, 38152-38158, October 12, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/41/38152    most recent M101980200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, L. Articles by Lotersztajn, S. Search for Related Content PubMed PubMed Citation Articles by Li, L. Articles by Lotersztajn, S. Social Bookmarking                      What's this?

15-Deoxy-^12,14-prostaglandin J2 Induces Apoptosis of Human Hepatic Myofibroblasts

A PATHWAY INVOLVING OXIDATIVE STRESS INDEPENDENTLY OF PEROXISOME-PROLIFERATOR-ACTIVATED RECEPTORS^*

Liying Li^§, Jiangchuan Tao^¶, Julien Davaille, Chloé Féral, Ariane Mallat, Jennifer Rieusset, Hubert Vidal, and Sophie Lotersztajn^**

From Unité INSERM 99, Hôpital Henri Mondor, 94010 Créteil, France and  INSERM U449, Faculté Alexis Carrel, 69372, Lyon Cedex 08, France

Received for publication, March 5, 2001, and in revised form, July 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-PGJ₂) 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-PGJ₂, was not expressed in hMFs. Moreover, 15-d-PGJ₂ 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-PGJ₂-treated hMFs exhibited condensed nuclei, fragmented DNA, and elevated caspase-3 activity. Moreover, the caspase inhibitor Z-Val-Ala-Asp(OCH₃)-fluoromethyl ketone blocked the cytotoxic effect of 15-d-PGJ₂. The apoptotic effects of 15-d-PGJ2 were reproduced by H₂O₂ and blocked by the antioxidants N-acetylcysteine (NAC), N-(2-mercapto-propionyl)-glycine (NMPG) and pyrrolidine dithiocarbamate (PDTC). Accordingly, 15-d-PGJ₂ generated rapid production of reactive oxygen species in hMFs, via a NAC/NMPG/PDTC-sensitive pathway. In conclusion, 15-d-PGJ₂ 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-PGJ₂.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Liver fibrosis is the common response to chronic liver injury, and it is characterized by increased deposition and altered composition of extracellular matrix (1). This fibrogenic process is consecutive to intense proliferation and hepatic accumulation of myofibroblasts that synthesize fibrosis components and proinflammatory cytokines (1). Several studies have highlighted the phenotypic heterogeneity of myofibroblasts, and Knittel et al. (2, 3) have characterized two subpopulations of fibrogenic cells, hepatic stellate cells and hepatic myofibroblasts.

Apoptosis of myofibroblasts is emerging as a key process in the regulation 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 myofibroblasts, followed by activation of fibrolysis mechanisms and regression of fibrosis (4). However, little is known regarding factors and mechanisms that regulate apoptosis of these cells. Thus, cultured hepatic stellate cells undergo apoptosis in response to soluble Fas (CD95/APO-1) ligand or nerve growth factor, by a pathway as yet uncharacterized (5-7). Apoptosis and growth inhibition are often governed by the same factors. We have recently demonstrated that cyclooxygenase-2 (COX-2),¹ the rate-limiting enzyme in the production of prostaglandins, plays a central role in growth inhibition of human hMFs (8-12). These findings raised the question as to whether COX-2-derived prostaglandins may induce hMF apoptosis.

Among COX-2-derived compounds, the cyclopentenone 15-deoxy-^12,14-prostaglandin J2 (15-d-PGJ₂) is recognized as a potent apoptotic and growth inhibitory factor (13, 14). 15-d-PGJ₂ is a downstream metabolite of PGD₂ and is produced by dehydration of PGD₂. In contrast to classical prostaglandins, which bind to cell surface G protein-coupled receptors, 15-d-PGJ₂ is a natural ligand of a nuclear receptor, the peroxisome-proliferator activated receptor  (PPAR) (13, 14). PPAR behaves as a ligand-activated transcription factor through its DNA binding domain, which recognizes response elements in the promoter of specific target genes linked to apoptosis, cell proliferation, differentiation, inflammation, and glucose homeostasis (13, 14).

In the present study, we show that the cyclopentenone prostaglandin 15-d-PGJ₂ elicits potent apoptotic effects in human hepatic myofibroblasts by a novel mechanism involving oxidative stress and unrelated to its nuclear receptor PPAR. These data underline the potential antifibrogenic effects of 15-d-PGJ₂.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 15-Deoxy-^12,14-prostaglandin J₂, ¹²-PGJ₂, PGJ₂, PGD₂, 15-deoxy-^12,14-prostaglandin A₂, PGA₂, and PGE₂ were obtained from Cayman (Alexis Corp.). Culture media and reagents were from Life Technologies, Inc. Fetal calf serum was from JBio Laboratories. Pooled human AB positive serum was supplied by the National Transfusion Center. WY-14643, ciglitazone, troglitazone, and N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (AC-DEVD-AFC) fluorogenic substrate were from Biomol (Tebu, France). The two sources of anti-PPAR antibodies used for Western blot were from Biomol and Santa Cruz Biotechnology (Tebu, France), and one of them (SC-6285) was also used in immunohistochemistry experiments. 2',7'-Dichlorohydrofluorescein diacetate (DCFH-DA) was from Molecular Probes (Interchim, Montluçon, France), and H₂O₂ and pyrrolidine dithiocarbamate (PDTC) were from Sigma. Z-Val-Ala-Asp(OCH₃)-fluoromethyl ketone (ZVAD-fmk) was from R&D Systems, 4',6'-diamidino-2-phenylindole (DAPI) was from Biovalley, CellTiter 96 AQueous One solution reagent was from Promega, and the cell death detection ELISA kit and apoptotic DNA ladder kit were from Roche Molecular Biochemicals. N-Acetylcysteine (NAC) and N-(2-mercapto-propionyl)-glycine (NMPG) (Sigma) were dissolved in PBS and buffered with NaOH to pH 7.4 prior to use. [³⁵S]GTPS was from ICN. Caco-2 cells were a generous gift of Dr. Marc Laburthe (INSERM U410, Paris, France).

Cell Isolation and Characterization-- Human hMFs were obtained by outgrowth of explants prepared from surgical specimens of normal liver, as previously described (15). This procedure was performed in accordance with ethical regulations imposed by French legislation. Cells were used between the third and seventh passage.

The myofibroblastic nature of the cells was routinely evaluated by electron microscopy and positivity for smooth muscle -actin by immunohistochemistry, as previously described (15). The cultures were also found to express two markers of rat hepatic myofibroblasts, fibulin-2 and interleukin-6, and not the protease P100, a marker for rat hepatic stellate cells (2).

Competitive Reverse Transcription and Amplification by Polymerase Chain Reaction for Human PPAR-- Total RNA was extracted from confluent quiescent hMFs using the RNeasy kit (Promega). The construction of the competitor DNA and the validation of the reverse transcription-competitive PCR for the different PPAR mRNAs have been described (16). The primers used for PPAR allowed the quantification of PPAR1 and PPAR2 (16). The reverse transcription-competitive PCRs and analysis of the PCR products were performed as described in Ref. 16.

Cell Viability-- Cells (7000 cells/well in 96-well plates) were allowed to attach overnight in DMEM containing 5% human serum and 5% fetal calf serum (DMEM 5/5), serum-starved for 48 h, and treated with the indicated effectors for 20 h. CellTiter 96 AQueous One Solution reagent was added to each well, and absorbance was recorded at 490 nm.

Apoptosis Assays-- All of the following techniques for measuring apoptosis were performed on nonconfluent cells allowed to attach overnight in DMEM 5/5 and serum-starved for 48 h. Nuclear morphology was assayed using DAPI staining. Cells (10,000/cm²) in Lab-Tek chamber slides (Nalge Nunc International) were treated with 15-d-PGJ₂ for 20 h, fixed in 2% paraformaldehyde, stained with DAPI, and viewed under fluorescence microscopy (Zeiss) using the blue filter. Quantification of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) was performed using a cell death detection ELISA kit. After treatment of cells (300,000 cells in 60-mm dishes) with the indicated effectors, attached and floating cells were collected and centrifuged. The supernatants were processed according to the manufacturer's instructions. Caspase-3-like activity was assayed on cell lysates obtained as follows. After treatment of cells (300,000 cells in 60-mm dishes) with the indicated effectors, floating cells were collected and centrifuged, and the pellet was lysed in 50 µl of lysis buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 1% Nonidet P-40, 1 mM EDTA (pH 8.0), 1 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin. Adherent cells were washed three times with cold PBS and lysed for 10 min on ice in 0.2 ml of lysis buffer. The lysates from adherent and floating cells were pooled and centrifuged, and the supernatant was stored at 80 °C until use. DEVDase activity was measured in 200 µl of assay buffer, containing 100 mM HEPES, pH 7.4, 10% sucrose, 10 mM dithiothreitol, 500 µM EDTA, 50 µg of protein, and 20 µM AC-DEVD-AFC as fluorogenic substrate. After 3 h at 37 °C, the fluorescence of the reaction mixture was determined with a spectrofluorometer (FL600 microplate fluorescence reader (Biotek Instruments), with excitation and emission wavelengths of 400 and 530 nm, respectively. DNA laddering was assayed by agarose gel electrophoresis of total DNA extracted from cells (800,000 cells in 100-mm dishes) treated for 20 h with the indicated effectors. Total DNA was extracted using the apoptotic DNA ladder kit according to the manufacturer's instructions and was further incubated with 20 µg/ml RNase (DNase-free) for 10 min at 30 °C. Two µg of DNA were electrophoresed on a 2% agarose gel stained with SYBR Green I and analyzed by PhosphorImager (Molecular Dynamics).

[³⁵S]GTPS Binding Assay-- Membranes were obtained from confluent hMFs made quiescent by incubation in Waymouth medium without serum for 48 h, as described in Ref. 17, and frozen at 80 °C until use. [³⁵S]GTPS binding was performed in the conditions described in Ref. 17.

Fluorescent Measurement of Intracellular Reactive Oxygen Species-- The fluorescent probe DCFH-DA (dissolved at 5 mM in absolute ethanol) was used for the assessment of intracellular reactive oxygen species (ROS). For microspectrofluorometry experiments, human hMFs were plated in 35-mm dishes (40,000 cells/dish), the bottoms of which were replaced by glass coverslips, and allowed to attach in DMEM 5/5 for 24 h. Cells were serum-deprived for 24 h in DMEM without phenol red, rinsed with PBS, and loaded for 40 min at 22 °C with 5 µM DCFH-DA in 2 ml of PBS. Unincorporated DCFH-DA was eliminated by two washes in PBS. DCFH-DA-loaded hMFs were placed on the stage of a Nikon diaphot inverted microscope with epifluorescence and further stimulated at 22 °C with H₂O₂ or 15-d-PGJ2. Fluorescence (Nikon UV-fluor × 20 objective) was measured using an excitation wavelength of 480 nm, and light from a 100-W xenon lamp was filtered through a 510-nm filter by an intensified charged-coupled device Photonic Science camera. Each fluorescence image was the average of 16 images, to improve the signal-to-noise ratio, and one average image was recorded every 3 s. Fluorescence intensity was assessed in region of interest (about 7 cells/field), and background was taken in cell-free regions. All tracings of fluorescence are representative of at least 20 cells and were performed on at least three different cell preparations.

When indicated, DCF fluorescence was measured using a FL-600 multiplate fluorometer (Biotek Instruments). Cells (7000 cells in 96-well plates) were allowed to attach overnight in DMEM 5/5 and serum-starved for 2 days. Cells were then loaded for 20 min at 37 °C with 5 µM DCFH-DA in PBS. After two washings in PBS, hMFs were incubated with either 15-d-PGJ₂ or H₂O₂, and the fluorescence was monitored after 5 min, using excitation and emission wavelengths of 485 and 530 nm, respectively. Values were corrected for hMF autofluorescence.

Preparation of Whole Cell, Nuclear, and Cytoplasmic Extracts-- Extracts were prepared from either confluent or nonconfluent quiescent hMFs and confluent Caco-2, as previously described (11). Adipose tissue proteins were prepared as described in Ref. 18.

Western Blotting Analysis-- Western blotting analysis was performed as previously described (17). Detection of PPAR was performed with two different sources of antibody after incubation for 2 h with antibody diluted 1:2000 for the Biomol source and 1:300 for the Santa Cruz Biotechnology source. Immunodetected proteins were visualized by using an enhanced chemiluminescence assay kit (Amersham Pharmacia Biotech).

Immunohistochemistry-- For immunocytochemistry, Caco-2 cells and hMFs (10,000 cells/cm²) were allowed to attach overnight on Lab-Tek slides, serum-starved for 2 days, and fixed for 7 min in 100% methanol at 20 °C. Cells were rinsed in PBS, incubated in PBS containing 1% bovine serum albumin (fraction V) for 1 h, and further incubated with anti-PPAR antibody (1:300 dilution) for 1 h. The cells were then washed three times with PBS, incubated for 1 h in the presence of Cy3-labeled rabbit anti-goat IgG (Sigma), rinsed three times with PBS, covered with Vectashield mounting medium containing DAPI, and viewed under fluorescence microscopy (Zeiss) using blue and red filters.

Statistics-- Results are expressed as mean ± S.E. of n experiments. Results were analyzed by repeated measures analysis of variance or two-tailed Student's t test, as appropriate, with p < 0.05 considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclopentenone Prostaglandins of the J Series Induce Apoptosis of Human hMFs-- We first assessed the effects of PGE₂, PGD₂, and their respective cyclopentenone derivatives of the J and A series, on the viability of human hMFs. As shown in Fig. 1, prostaglandins of the J series strongly reduced hMF viability, whereas PGD₂ was cytotoxic at higher concentrations. 15-d-PGJ₂ was the most potent compound, followed by its precursors ¹²PGJ₂ and PGJ₂. Among the prostaglandins of the A series, only PGA₂ reduced hMF viability, whereas its precursor PGE₂ and its metabolite 15-d-PGA₂ had no effect.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Cyclopentenone prostaglandins of the J series decrease hMF viability. Serum-deprived cells were incubated with 5 µM 15-d-PGJ2, 50 µM PGJ2, and 100 µM PGD2 for the indicated periods of time (A) and with varying concentrations of 15-d-PGJ2, 12-PGJ2, PGJ2, 15-d-PGA2, PGA2, and PGE2 for 20 h (B). Cell viability was determined as described under "Experimental Procedures." Results are the mean ± S.E. of four experiments.

In subsequent experiments, we investigated the mechanism of action of the prostaglandin with the most potent cytotoxic effect and focused on 15-d-PGJ₂.

The cyclopentenone prostaglandin 15-d-PGJ₂ strongly reduced hMF viability, as assessed by phase contrast analysis. Indeed, 15-d-PGJ₂ caused shrinkage, rounding and detachment of hMFs, whereas serum deprivation alone (control) did not affect the morphology of the cells (Fig. 2A). Addition of 5% human serum totally protected human hMFs from the cytotoxic effect of 15-d-PGJ₂ (not shown). Further experiments indicated that apoptosis was responsible for cell death. DAPI staining showed that 90% of the nuclei exhibited condensed nuclei after 20 h of treatment with 5-10 µM 15-d-PGJ₂, whereas serum-deprived cells exhibited normal nuclei (Fig. 2B). Consistently, 15-d-PGJ₂-treated cells showed dramatic DNA laddering on agarose gel electrophoresis, whereas control serum-deprived cells exhibited intact DNA (Fig. 2C). ELISA detection of histone-bound DNA fragments indicated that the apoptotic effect of 15-d-PGJ₂ was time-dependent, with a maximal effect observed after 16 h of exposure to the prostaglandin (Fig. 2D). To further characterize the apoptotic effect of 15-d-PGJ₂ on hMFs, we used the general caspase inhibitor ZVAD-fmk. As shown in Fig. 2E, ZVAD-fmk blunted 15-d-PGJ₂-induced cell death. Accordingly, 15-d-PGJ₂ caused a potent time-dependent activation of caspase-3-like activity, as assessed by DEVDase activity. Maximal activation occurred after 8-10 h stimulation and was totally blunted by ZVAD-fmk (Fig. 2F).

View larger version (78K): [in this window] [in a new window]   Fig. 2.   Apoptotic effects of 15-d-PGJ2 in human hMFs. A, phase contrast analysis (× 100). Serum-deprived cells were cultured for 20 h in Waymouth medium in the presence or absence of 8 µM 15d-PGJ2. B, DAPI staining of the nuclei (× 680). Serum-deprived cells were incubated for 20 h in the presence of 8 µM 15d-PGJ2 or vehicle, fixed, and stained with DAPI. C, DNA ladder formation. Cells were treated as in A, and DNA was extracted and further analyzed by electrophoresis on a 2% agarose gel stained with SYBR green. Left lane, 1-kilobase pair DNA ladder. D, ELISA detection of histone-bound DNA fragments. Serum-deprived cells were incubated for the indicated periods of time with 8 µM 15d-PGJ2 or vehicle. ELISA detection of histone-bound DNA fragments was performed as described under "Experimental Procedures." Results are expressed as percentage of untreated controls and are the mean ± S.E. of three experiments. E, 15-d-PGJ2-induced cell death is a caspase-dependent process. Serum-deprived cells were preincubated for 1 h with 50 µM ZVAD-fmk or vehicle and further incubated for 20 h with varying concentrations of 15-d-PGJ2. Cell viability was determined as described under "Experimental Procedures." Results are the mean ± S.E. of four experiments (p < 0.01 for 15d-PGJ2 + Z-VAD-fmk versus 15d-PGJ2 alone). F, caspase-3-like activity. Serum-deprived cells were preincubated for 1 h with 50 µM ZVAD-fmk or vehicle and further incubated with 8 µM 15d-PGJ2 or vehicle for the indicated periods of time. Caspase-3-like activity was assayed on lysates using the fluorogenic substrate AC-DEVD-AFC. Results represent the mean ± S.E. of triplicate determinations. A typical experiment repeated twice is shown. DEVDase activity is expressed as pmol/mg of protein/min (p < 0.01 for 15d-PGJ2 + Z-VAD-fmk versus 15d-PGJ2 alone).

Taken together, these data demonstrate that 15-d-PGJ₂ induces caspase-dependent apoptosis of human hMFs.

The Apoptotic Effect of 15-d-PGJ₂ Does Not Involve Either PPAR or a G Protein-coupled Receptor-- Because cyclopentenone prostaglandins of the J series, and in particular 15-d-PGJ₂, are potent PPAR ligands (13, 14), we studied the effects of pharmacological PPAR agonists on hMF viability. Surprisingly, ciglitazone or troglitazone did not induce hMF cell death (Fig. 3A). As 15-d-PGJ₂ is also a ligand of PPAR at very high concentrations (>10 µM) (13, 14), we also assessed the effects of a PPAR agonist, WY 14643. At doses reported to fully activate PPAR, WY 14643 did not affect cell viability (Fig. 3A) (13, 14). We then investigated PPAR expression in human hMFs by quantitative reverse transcription-PCR, Western blot analysis, and immunohistochemistry. Human hMFs did not express PPAR mRNA (Fig. 3B), nor could we detect the corresponding PPAR1 and PPAR2 proteins (Fig. 3, C and D), using two different sources of antibody, in either confluent or subconfluent cells or in nuclear or whole cell lysate. In contrast, and as expected, PPAR was expressed in human adipose tissue and Caco-2 cells, both used as controls (Fig. 3, C and D). Human hMFs expressed low levels of PPAR and PPAR mRNAs (0.6 ± 0.4 and 0.8 ± 0.2 atomoles/µg of total RNA, for PPAR and PPAR, respectively; n = 4) (Fig. 3B), as compared with total human liver (17 ± 1.6 and 2.2 ± 0.7 atomoles/µg total RNA for PPAR and PPAR, respectively; data not shown, but see Ref. 16).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   15-d-PGJ2 induces apoptosis of human hMFs independently of PPAR expression. A, cell viability. Serum-deprived cells were incubated for 20 h with varying concentrations of 15-d-PGJ2, ciglitazone, troglitazone, and WY14643. Cell viability was determined as described under "Experimental Procedures." Results are the mean ± S.E. of three experiments. B, characterization of PPAR mRNA expression by quantitative reverse transcription-PCR. C, Western blot analysis of PPAR expression was performed using two distinct antibodies (Santa Cruz Biotechnology (top) and Biomol (bottom)). For whole cell (confluent or nonconfluent) and nuclear extracts of human hMFs, as well as for Caco-2 cells, 40 µg of protein were loaded. For adipose tissue, 1 µg of protein was loaded. D, immunocytochemical detection of PPAR expression in human hMFs (c and d) and Caco-2 cells (a and b). a and c show the signal obtained when the first antibody was omitted.

Given the absence of PPAR in human hMFs, we explored the possibility that 15-d-PGJ2 binds to a G protein-coupled receptor, as described for other prostanoids. We therefore performed [³⁵S]GTPS binding assays, which measure GDP-GTP exchange on the  subunit of the G protein and reflect the initial steps of G protein activation by a receptor ligand. Optimal binding conditions were defined with classical ligands of G protein-coupled receptors, such as sphingosine-1-phosphate, PGE₂, or carbacyclin, the stable analog of PGI₂, all of which stimulated [³⁵S]GTPS binding (Fig. 4, inset). In contrast, 15-d-PGJ₂ did not increase [³⁵S]GTPS binding, even when varying GDP concentrations were added to keep the G protein in the nondissociated form (Fig. 4). These results indicate that 15-d-PGJ₂ does not bind to a G protein-coupled receptor in human hMFs.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Absence of effect of 15-d-PGJ2 on GTPS binding in human hMFs. [35S]GTPS binding was assayed in membranes from human hMFs, as described under "Experimental Procedures," with varying concentrations of GDP, in the presence of 10 µM 15-d-PGJ2 or vehicle. Results represent a typical experiment that was repeated twice and are expressed as percent of control. Inset, GTPS binding was assayed with 10 µM GDP in the presence of a 30 µM concentration of either carbacyclin (c-PGI2), PGE2, PGA2, or 10 µM sphingosine-1-phosphate (S1P). Results are the mean ± S.E. of three experiments.

Altogether, these data suggest that 15-d-PGJ₂ induces cell death of human hMFs by a mechanism unrelated to the nuclear receptor PPAR or to a G protein-coupled receptor.

The Apoptotic Effect of 15-d-PGJ₂ Involves Production of Reactive Oxygen Species-- Cyclopentenone prostaglandins may activate the stress response by inducing several stress-related genes (19-21). Therefore, we examined whether cell death induced by 15-d-PGJ₂ involves oxidative stress. Exogenously added H₂O₂ induced cell death by an apoptotic process, as indicated by DNA fragmentation (Fig. 5A) and time-dependent caspase-3-like activation (Fig. 5B). Accordingly, the caspase inhibitor ZVAD-fmk blunted H₂O₂-induced cytotoxicity (Fig. 5C).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Apoptotic effects of H2O2. A, DNA ladder formation. DNA was extracted from serum-deprived hMFs treated for 20 h with 300 µM H2O2 and analyzed as in Fig. 2C. B, caspase-3-like activity. Serum-deprived cells were preincubated for 1 h with 50 µM ZVAD-fmk or vehicle and further incubated with 300 µM H2O2 or vehicle for the indicated times. Results represent the mean ± S.E. of triplicate determinations. A typical experiment repeated twice is shown. DEVDase activity was measured as described in Fig. 3F. C, H2O2-induced cell death is a caspase-dependent process. Serum-deprived cells were preincubated for 1 h with 50 µM ZVAD-fmk or vehicle and further incubated with H2O2 or vehicle for 20 h. Cell viability was measured as described in Fig. 2E. Results are the mean ± S.E. of four experiments (*, p < 0.01 versus H2O2 alone).

We next assessed the effect of several antioxidants on the apoptotic effects of 15-d-PGJ₂. NAC, NMPG, and PDTC blunted 15-d-PGJ₂-induced activation of caspase-3-like activity (Fig. 6A) and DNA fragmentation (Fig. 6B). As expected, NAC, NMPG, and PDTC also blunted caspase-3-like activation by H₂O₂ (Fig. 6A, inset). To further support the role of ROS in the apoptotic effect of 15-d-PGJ₂, we investigated whether 15-d-PGJ₂ produces ROS in hMFs.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   15-d-PGJ2-induced apoptosis involves ROS production. Serum-deprived hMFs were pretreated for 1 h with either 5 mM NAC, 5 mM NMPG, 0.1 mM PDTC, or vehicle. A, caspase-3-like activity was assayed as in Fig. 3, after a 10-h treatment with 8 µM 15d-PGJ2, 300 µM H2O2 (inset) or vehicle (*, p < 0.01 versus control; #, p < 0.01 versus 15-d-PGJ2 or H2O2 (inset) alone). Results represent the mean S.E. of three experiments. B, DNA ladder formation was analyzed as in Fig. 3, after a 20-h treatment with 8 µM 15d-PGJ2 or vehicle. Antioxidants added alone had no effect on caspase-3-like activity or DNA ladder formation.

15-d-PGJ₂ Increases Intracellular Reactive Oxygen Species in Human hMFs-- The formation of intracellular ROS was assessed using the peroxide-sensitive probe DCFH-DA. This probe diffuses through the cell membrane and is hydrolyzed to dichlorohydrofluorescein (DCFH), following cleavage of the diacetate group by intracellular esterases. In the presence of ROS, DCFH is rapidly oxidized to highly fluorescent dichlorofluorescein (DCF). As shown by microspectrofluorometry, 20 µM H₂O₂ caused a rapid and marked increase in DCF fluorescence after 0.5-2 min (Fig. 7A). Addition of 15-d-PGJ₂ also produced a rapid and dose-dependent increase in DCF fluorescence within 5-10 min. A small increase in fluorescence was observed with 1 µM 15-d-PGJ₂ in 40% of cells (3 responding cells out of 8), whereas 70% (69 cells out of 92) and 100% (40 cells out of 40) of cells responded to 5 and 10 µM prostaglandin, respectively (Fig. 7B). The fluorescence peak increased sharply in 70% of cells treated with 2.5 or 5 µM 15-d-PGJ₂ and smoothly in the remaining 30% responding cells.

View larger version (64K): [in this window] [in a new window]   Fig. 7.   Effect of H2O2 and 15-d-PGJ2 on the production of ROS in human hMFs. hMFs were loaded with DCFH-DA and further stimulated with 20 µM H2O2 (A) or with varying concentrations of 15-d-PGJ2 (B). Fluorescence was measured using microspectrofluorometry. On the left are shown the pseudocolored images obtained after 2 min of incubation with PBS and after either 6 min of stimulation with H2O2 or 10 min of incubation with 15-d-PGJ2. On the right is shown a representative trace of fluorescence, obtained from the cell indicated by the arrows.

Fluorometry was used to further quantify ROS production in response to 15-d-PGJ₂. 15-d-PGJ₂ caused a 2.5- and 3.2-fold increase in DCF fluorescence at 5 and 10 µM, respectively (Fig. 8). The antioxidants NAC, NMPG, and PDTC strongly reduced ROS production elicited by 15-d-PGJ₂, whereas L-N^G-Nitroarginine-methyl ester had no effect (Fig. 8). These results suggest that variations in DCF fluorescence induced by 15-d-PGJ₂ are indeed related to superoxide or hydrogen peroxide formation and not to nitric oxide production. Taken together, these results demonstrate that 15-d-PGJ₂ induces apoptosis of hMFs by a mechanism involving oxidative stress.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Effect of antioxidants on the production of ROS promoted by 15-d-PGJ2 in human hMFs. hMFs were preincubated for 60 min with either 5 mM NAC, 5 mM NMPG, 0.1 mM PDTC, 2 mM L-NG-Nitroarginine methyl ester, or vehicle and then loaded with DCFH-DA for 20 min at 37 °C. After two washings, 15-d-PGJ2 was added, and the fluorescence was monitored in a FL-600 fluorometer. Results are the mean ± S.E. of at least three experiments. #, p < 0.05 for 15-d-PGJ2 versus basal. *, p < 0.05 versus the respective concentration of 15-d-PGJ2 alone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We show here that 15-d-PGJ₂ is a potent apoptotic factor for human hepatic myofibroblasts and may therefore act as an inhibitor of liver fibrogenesis. Importantly, 15-d-PGJ₂-induced cell death is independent of PPAR activation and involves oxidative stress.

Myofibroblasts play a central role in the development of liver fibrosis. During this process, they proliferate, accumulate in the diseased liver, and secrete fibrosis components. Therefore, blockade of hMF proliferation and enhancement of hMF apoptosis are emerging as potential therapeutic goals. We previously reported the growth inhibitory properties of endothelin-1, tumor necrosis factor , C-type natriuretic peptide, and sphingosine-1-phosphate in human hMFs (11, 12, 17, 22) and demonstrated the critical role of COX-2-derived prostaglandins in this process (9, 11, 12). We show here that 15-d-PGJ₂ is a potent apoptotic factor for hepatic myofibroblasts, based on several lines of evidence (Fig. 2). First, 15-d-PGJ₂ induces hMF rounding and detachment. Second, 15-d-PGJ₂-treated hMFs exhibit condensed nuclei upon DAPI staining. Third, hMFs undergo DNA fragmentation, as shown by gel electrophoresis and quantification of histone-bound DNA fragment. Finally, 15-d-PGJ₂ stimulates caspase-3-like activity, prior to loss of hMF viability, and the caspase inhibitor ZVAD-fmk blunts 15-d-PGJ₂-induced cell death. Overall, these features are the hallmarks of programmed cell death. Our results therefore identify 15-d-PGJ₂ as a novel apoptotic stimulus for human hepatic myofibroblasts. Other factors that trigger apoptosis of liver fibrogenic cells have been described in rat and include nerve growth factor, Fas ligand, and serum deprivation (9, 11, 12). However, these studies did not evaluate the early signaling pathways involved in the apoptotic process.

Prostaglandins of the J series, and more specifically 15-d-PGJ₂, exhibit several functions, including growth arrest, apoptosis, differentiation, and suppression of macrophage activation and inflammation (13). It has recently been shown that 15-d-PGJ₂ is an endogenous ligand for the nuclear receptor PPAR, which is currently considered a major receptor for these prostaglandins (13). However, our results indicate that in human hMFs, 15-d-PGJ₂-induced apoptosis is unrelated to PPAR (Fig. 3), because PPAR is not expressed in these cells, in keeping with the absence or limited expression of PPAR in myofibroblastic hepatic stellate cells (23-25). In addition, classical ligands of PPAR or PPAR do not affect the viability of hMFs. Dissociation between the biological effects of 15-d-PGJ₂ and those mediated by PPAR has been described in a few instances (26-29). However, evidence for the existence of a specific cell surface prostanoid receptor for 15-d-PGJ₂ is sketchy. In cells overexpressing the human receptor for PGD₂ (DP receptor), 15-d-PGJ₂ displaces PGD₂ binding, suggesting its binding to the DP receptor (30). We used [³⁵S]GTPS binding assays, which reflect the initial steps of G protein activation by a G protein-coupled receptor ligand, to test the hypothesis of a cell surface receptor for 15-d-PGJ₂. We show that 15-d-PGJ₂ does not stimulate [³⁵S]GTPS binding assays in human hMFs (Fig. 4). Therefore, the presence of a specific G protein-coupled receptor for 15-d-PGJ₂ seems unlikely. An alternate hypothesis could be that in hMFs, 15-d-PGJ₂-induced apoptosis is associated with a receptor-independent active transport of the prostaglandin, followed by its accumulation into the nucleus and endoplasmic reticulum, as described in kidney and leukemia cells (19, 31).

Apoptotic signaling pathways of 15-d-PGJ₂ in other cells are poorly understood. It has been reported that 15-d-PGJ₂ inhibits NF-B by directly blunting IB kinase and may therefore block NF-B-dependent antiapoptotic gene expression (26-29). However, 15-d-PGJ₂ had no effect on IB- degradation by tumor necrosis factor  in human hMFs (not shown). A major point of the present study is that generation of reactive oxygen species serves as second messenger of the apoptotic effect of 15-d-PGJ₂. Indeed, exogenously added H₂O₂ induces hMF apoptosis, as shown by its cytotoxic effect via a caspase-dependent process and by the induction of DNA laddering (Fig. 5). Moreover, the antioxidants NAC, NMPG, and PDTC decrease the apoptotic response to 15-d-PGJ₂ (Fig. 6). Finally, 15-d-PGJ₂ stimulates ROS production, as shown by measuring oxidation of the peroxide-sensitive fluorescent probe DCFH-DA (Figs. 7 and 8).² Production of ROS is rapid, being observed after 5-10 min of stimulation with 15-d-PGJ₂, and therefore appears as an early signaling event in the apoptotic signaling pathway. ROS production is sensitive to NAC, NMPG, and PDTC and is unaffected by inhibitors of nitric oxide synthesis, indicating that 15-d-PGJ₂ promotes superoxide or hydrogen peroxide formation rather than nitric oxide production. It should be noted that extracellularly added superoxide dismutase and catalase did not protect from 15-d-PGJ₂-induced ROS production and hMF death. Similar results have been obtained in sarcoma cells exposed to hepatocyte growth factor (32). Possible explanations for inability of superoxide dismutase and catalase to prevent 15-d-PGJ₂-induced hMF death include limited transport of these enzymes into human hMFs and inaccessibility to the intracellular compartment responsible for 15-d-PGJ₂-stimulated ROS production. Increasing evidence suggests a major role for ROS as intermediates for apoptosis signaling. Thus, production of ROS leads to growth inhibition and apoptosis of tumor and hematopoietic cells in response to hepatocyte growth factor, tumor necrosis factor , or Fas ligand (32, 33). The signaling events initiated by ROS following 15-d-PGJ₂ stimulation and leading to human hMF apoptosis are under current investigation.

Apoptotic and growth inhibitory properties of 15-d-PGJ₂ in human hMFs suggest that this cyclopentenone prostaglandin may be a negative regulator of liver fibrogenesis. Whether liver cells may produce 15-d-PGJ₂ is currently unknown. 15-d-PGJ₂ may derive from PGD₂, the most abundant prostaglandin in normal tissue, and its metabolite 12-PGJ₂, the precursor of 15-d-PGJ₂, is present in body fluids (34). In addition, elevated 15-d-PGJ₂ levels were detected in the extracellular fluid of inflammatory exudates (35). Also, PGJ₂-like compounds are generated in the liver of normal rats and increase dramatically in the liver following acute treatment with CCl4 (36). Among liver cells, Kupffer cells and hepatic myofibroblasts are potential sources of 15-d-PGJ₂, because both cell types display high levels of COX-2 activity and may release PGD₂ (11, 37). We are currently evaluating the antifibrogenic potential of 15-d-PGJ₂.

	ACKNOWLEDGEMENTS

We thank J. Hanoune for constant support and F. Pecker, Y. Laperche, and G. Guellaen for critical reading of the manuscript. We thank C. Pavoine for constant support during microspectrofluorometry experiments and for helpful discussions. We acknowledge J. P. Riou for helpful suggestions and Marc Laburthe for the kind gift of Caco-2 cells.

	FOOTNOTES

^* This work was supported by INSERM, by the Université Paris-Val-de-Marne, and by grants from the Association pour la Recherche sur le Cancer (to S. L.) and from the Ligue Départementale du Val d'Oise de la Recherche contre le Cancer (to S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Contributed equally to this work.

^§ Supported by an INSERM fellowship.

^¶ Supported by a fellowship of the Fondation pour la Recherche Médicale.

^** To whom correspondence should be addressed. Tel.: 33-1-49-81-35-34; Fax: 33-1-48-98-09-08; E-mail: loterszt@im3.inserm.fr.

Published, JBC Papers in Press, July 27, 2001, DOI 10.1074/jbc.M101980200

² While this paper was under review, Kondo et al. (38) demonstrated that 15-d-PGJ₂ increases intracellular stress in SH-SY5Y human neuroblastoma cells.

	ABBREVIATIONS

The abbreviations used are: COX, cyclooxygenase; AC-DEVD-AFC, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; DAPI, 4',6'-diamidino-2-phenylindole; DCF, dichlorofluorescein; DCFH, dichlorohydrofluorescein; DCFH-DA, 2',7'-dichlorohydrofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; GTPS, guanosine-5'-O-(3-thiotriphosphate; hMF, hepatic myofibroblast; NAC, N-acetylcysteine; NMPG, N-(2-mercapto-propionyl)-glycine; ROS, reactive oxygen species; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDTC, pyrrolidine dithiocarbamate; PG, prostaglandin; PPAR, peroxisome-proliferator-activated receptor; ZVAD-fmk, Z-Val-Ala-Asp(OCH₃)-fluoromethyl ketone; 15-d-PGJ₂, 15-deoxy-^12,14-prostaglandin J₂.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				W. I. Hagens, L. Beljaars, D.A. Mann, M. C. Wright, B. Julien, S. Lotersztajn, C. Reker-Smit, and K. Poelstra Cellular Targeting of the Apoptosis-Inducing Compound Gliotoxin to Fibrotic Rat Livers J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 902 - 910. [Abstract] [Full Text] [PDF]

				V. Serriere-Lanneau, F. Teixeira-Clerc, L. Li, M. Schippers, W. de Wries, B. Julien, J. Tran-Van-Nhieu, S. Manin, K. Poelstra, J. Chun, et al. The sphingosine 1-phosphate receptor S1P2 triggers hepatic wound healing FASEB J, July 1, 2007; 21(9): 2005 - 2013. [Abstract] [Full Text] [PDF]

				K. STAMATAKIS and D. PEREZ-SALA Prostanoids with Cyclopentenone Structure as Tools for the Characterization of Electrophilic Lipid-Protein Interactomes Ann. N.Y. Acad. Sci., December 1, 2006; 1091(1): 548 - 570. [Abstract] [Full Text] [PDF]

				P. B. Cassidy, K. Edes, C. C. Nelson, K. Parsawar, F.A. Fitzpatrick, and P. J. Moos Thioredoxin reductase is required for the inactivation of tumor suppressor p53 and for apoptosis induced by endogenous electrophiles Carcinogenesis, December 1, 2006; 27(12): 2538 - 2549. [Abstract] [Full Text] [PDF]

				D. M. Ray, F. Akbiyik, and R. P. Phipps The Peroxisome Proliferator-Activated Receptor {gamma} (PPAR{gamma}) Ligands 15-Deoxy-{Delta}12,14-Prostaglandin J2 and Ciglitazone Induce Human B Lymphocyte and B Cell Lymphoma Apoptosis by PPAR{gamma}-Independent Mechanisms J. Immunol., October 15, 2006; 177(8): 5068 - 5076. [Abstract] [Full Text] [PDF]

				E Novo, F Marra, E Zamara, L Valfre di Bonzo, L Monitillo, S Cannito, I Petrai, A Mazzocca, A Bonacchi, R S M De Franco, et al. Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans Gut, August 1, 2006; 55(8): 1174 - 1182. [Abstract] [Full Text] [PDF]

				F Marra Thiazolidinediones and hepatic fibrosis: don't wait too long Gut, July 1, 2006; 55(7): 917 - 919. [Full Text] [PDF]

				J. Pandhare, S. K. Cooper, and J. M. Phang Proline Oxidase, a Proapoptotic Gene, Is Induced by Troglitazone: EVIDENCE FOR BOTH PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {gamma}-DEPENDENT AND -INDEPENDENT MECHANISMS J. Biol. Chem., January 27, 2006; 281(4): 2044 - 2052. [Abstract] [Full Text] [PDF]

Z. Y. Chen and C.-C. Tseng 15-Deoxy-{Delta}12,14 Prostaglandin J2 Up-Regulates Kruppel-Like Factor 4 Expression Independently of Peroxisome Proliferator-Activated Receptor {gamma} by Activating the Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signal Transduction Pathway in HT-29 Colon Cancer Cells Mol. Pharmacol., November 1, 2005; 68(5): 1203 - 1213. [Abstract]