INTRODUCTION

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Hepatic stellate cells (1) (HSC,¹ also known as lipocytes, fat-storing cells, or perisinusoidal cells) play a crucial role in the pathogenesis of liver inflammation and fibrosis (1). Following acute or chronic liver injury, HSC transdifferentiate from a quiescent cell containing large retinoid droplets to an activated myofibroblast-like cell (2). In response to growth factors and cytokines expressed by neighboring cells, activated HSC display enhanced mitogenicity and secrete multiple inflammatory cytokines and components of fibrosis (1). Experimental models of liver injury have shown an increased proliferation of activated HSC (3, 4), and several studies have focused on factors that may limit growth of these cells. In particular, we have shown that binding of endothelin-1 (ET-1) to its G-protein-coupled ETB receptor leads to inhibition of human activated myofibroblastic HSC proliferation, following prostaglandin production (5, 6).

NF-B is an essential regulator of the expression of viral genes, as well as of a number of genes involved in immune, inflammatory, and growth responses (7-9). This family of transcription factors comprise at least five members, p65 (RelA), p50, p52, RelB, and the product of the c-Rel protooncogene, which form homo- or heterodimers and bind to a DNA sequence called the B motif. In most cell types, NF-B is present in the cytosol in an inactive form, as a heterodimer of a 50-kDa (p50) and 65-kDa (p65) subunit, bound to one of the IB inhibitory proteins. Of the different IBs, IB- and the more recently identified IB- have been well characterized. All known activators of NF-B (cytokines, phorbol esters, growth factors, and viral trans-activators) rapidly phosphorylate IB-, resulting in its ubiquitination and degradation through a proteasome-dependent pathway. Degradation of IB- occurs more slowly and is induced by agonists that cause persistent activation of NF-B (10). The free NF-B dimer subsequently translocates into the nucleus, where it activates gene transcription (7-9, 11). Resynthesis of IB- is induced by NF-B and allows resequestration of NF-B in the cytoplasm, shutting down the NF-B response (11).

Cyclooxygenases (COX) are the rate-limiting enzymes in the biosynthetic pathway of prostaglandins and thromboxanes from arachidonic acid. These enzymes catalyze the conversion of arachidonic acid to PGH2, the committed step in prostanoid synthesis. Two isozymes are found in mammalian tissues, COX-1 and COX-2. The COX-1 isoform is constitutively expressed in most cells, and the COX-2 gene, generally absent in resting cells, is rapidly induced by hormones, cytokines, and tumor promoters (12). Sequencing of the human cyclooxygenase-2 promoter region has revealed the presence of two NF-B consensus sites, which are important in the induction of the COX-2 mRNA by TNF- (13).

Since some evidence supports a role of NF-B in the activation of genes that regulate cell growth arrest (14-16), in this study we investigated whether activation of NF-B may be involved in the negative control of human myofibroblastic HSC proliferation. We show that two factors that inhibit the growth of human HSC, ET-1 (5, 6) and TNF- (the present study), activate NF-B in these cells. We also report that activation of NF-B by both factors leads to the induction of cyclooxygenase-2 (COX-2), which mediates inhibition of human HSC growth. All these results suggest that NF-B, by inducing COX-2 expression, may play an important role in the negative regulation of human myofibroblastic HSC proliferation.

	EXPERIMENTAL PROCEDURES

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Materials-- ET-1 and sarafotoxin S6C were from Novabiochem (France), and TNF- was from Preprotech (Tebu, France). Sodium salicylate, dexamethasone, and ibuprofen were obtained from Sigma (France). [methyl-³H]Thymidine (25 Ci/mmol) and [-³²P]ATP (5,000 Ci/mmol) were purchased from Amersham Corp. (France). Fetal calf serum was from J Bio Laboratories (France). Pooled human AB positive serum was supplied by the National Transfusion Center. Antibodies against p65 and IB- were from Santa Cruz Biotechnology (Tebu, France), and antibodies against p50 and IB- were kindly provided by Robert Weil (URA CNRS 1149, Paris, France). SC-58125 was a gift from Dr. P. C. Isakson (Searle). MG-132 was from Biomol (Tebu, France). cDNA probe for human COX-2 was a gift from Dr. S. Prescott (University of Utah), and the human G3PDH cDNA probe was from CLONTECH (Ozyme, France).

Isolation and Culture of Human Myofibroblastic HSC-- Human HSC in their activated myofibroblastic phenotype were obtained by outgrowth of normal liver explants obtained from surgery of benign or malignant liver tumors. This procedure was performed in accordance with ethical regulations imposed by the French legislation. Explants were incubated in Dulbecco's modified Eagle's medium containing 10% serum (5% fetal calf serum, 5% pooled human serum), and exhaustive characterization of these cells has already been published (17). Cell isolates were routinely characterized by a positive staining for smooth muscle -actin, a marker of HSC in their myofibroblastic phenotype. Experiments were performed between passages 3 and 7, without any noticeable difference in results observed with cells obtained from various passages or from various livers. All experiments were performed on confluent cells made quiescent in serum-free Waymouth medium over 3 days.

DNA Synthesis Assay-- DNA synthesis was measured in triplicate wells by incorporation of [³H]thymidine, as described previously (6). Following pretreatment for 60 min with 25 µM SC-58125 or 18 h with dexamethasone or vehicle, confluent quiescent cells were stimulated for 30 h with TNF- or ET-1, in the presence of 5% human serum. [³H]Thymidine (0.5 µCi/well) was added during the last 6 h of incubation.

Preparation of Whole Cell and Nuclear and Cytoplasmic Extracts-- Confluent cells in 12-well plates were made quiescent in serum-free Waymouth medium over 3 days. HSC were then incubated for various times with either ET-1 (0.1 µM), sarafotoxin S6C (0.1 µM), TNF- (50 ng/ml), or vehicle. When indicated, cells were pretreated either for 30 min with the NF-B inhibitors sodium salicylate (20 mM), or MG-132 (50 µM), or 18 h with dexamethasone (0.1 µM). After a wash in ice-cold phosphate-buffered saline, cells were lysed for 15 min at 4 °C in whole cell extraction buffer (50 mM Hepes, pH 7.4, containing 0.5% Nonidet P-40, 10% glycerol, 137 mM NaCl, 1 mM EGTA, pH 8, 10 mM NaF, 1 mM vanadate, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 40 mM -glycerophosphate, 0.1 mM DTT). Lysates were centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatants (whole cell extract) were stored at 80 °C until use.

Preparation of DNA Probes for Electrophoretic Mobility Shift Assay-- Sense and antisense oligomers containing NF-B-binding sites were annealed, and the double-stranded oligomer was radiolabeled with T4 polynucleotide kinase and [-³²P]ATP. The unincorporated nucleotides were removed by filtration though a G50 Fine column. Sequences of the oligomers containing the positive regulatory element (PRE) B sequence of the HIV-LTR were 5'-ACAAGGGACTTTCCGCTGGGGACTTTCCAGG-3', and oligomers containing the B-responsive element of the COX-2 promoter were 5'-GGAGAGGGGATTCCCTGCGC-3'. In some experiments, we also used with similar results an oligomer (Promega) corresponding to the consensus sequence of NF-B from the  light chain enhancer. The sequence of the oligomer containing the HNF3 sequence of the HNF1 promoter was 5'-TCGAGCTCCAATGTAAACAGAGCAGGT-3'.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts (5-10 µg of protein) were incubated in the binding reaction medium (20 mM Hepes, pH 7.9, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) for 15 min at 4 °C, followed by a 15-min incubation with 0.5 ng of ³²P-labeled oligonucleotide at room temperature. The DNA-protein complexes were analyzed on a 5% polyacrylamide gel in 0.25× Tris borate/EDTA electrophoresis buffer. Gels were run at 150 V for 90 min, dried, and autoradiographed. In competition assays, 100 ng of either unlabeled NF-B, HNF3, or COX-2 oligonucleotides were added to the binding reaction for 15 min before addition of the radiolabeled probe. In the supershift experiments, the binding reaction containing the antibodies were incubated for 1 h at room temperature before adding the radioactive probe.

RNA Preparation and Northern Blot Analysis-- Confluent cells were made quiescent in serum-free Waymouth medium over 3 days and were then stimulated with 0.1 µM ET-1 or 50 ng/ml TNF-, as indicated. Total RNA was extracted in guanidinium thiocyanate, as described previously (5). RNA samples (20 µg/lane) were denatured, fractionated by electrophoresis through a 1% agarose/formaldehyde gel, and subsequently transferred to a nitrocellulose membrane (Schleicher & Schuell). Prehybridization was performed at 42 °C in 5× SSC (1×SSC = 150 mM NaCl, 15 mM sodium citrate) containing 50% formamide, 1× Denhardt's, 20 mM NaH₂PO₄, 10% dextran, 170 µg/ml heparin, and 200 µg/ml salmon sperm DNA. cDNA probes for COX-2 and human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were labeled with [-³²P]dATP using a random priming Ready to Go kit (Pharmacia). Blots were hybridized overnight at 42 °C with the ³²P-labeled probes, washed at 22 °C for 1 min with 2× SSC, 0.1% SDS and once in 0.2× SSC, 0.1% SDS at 55 °C and exposed to x-ray film (Hyperfilm, Amersham) at 70 °C. Hybridization signals were quantified by Fuji Bio-imaging analyzer (Fuji, Tokyo, Japan). Results are relative to G3PDH expression, which was used as an internal standard to correct for variations in loading and transfer. Results are expressed as fold over basal COX-2/G3PDH ratio.

Western Blotting Analysis-- After electrophoresis on a denaturing SDS gel, proteins were electroblotted onto nitrocellulose membranes and blocked for 1 h at room temperature in 10 mM Tris, pH 8, containing 150 mM NaCl, 0.05% Tween 20, 5% skim milk. Detection of IB-, IB- (19), COX-1 (20), and COX-2 (21) was performed after incubation for 2 h at room temperature, with their specific antibody diluted 1:1000, 1:500, 1:2000, and 1:5000-fold, respectively. After 1 h incubation with 2000-fold diluted horseradish peroxidase-conjugated anti-IgG antibody and extensive washings, immunodetected proteins were visualized by using an enhanced chemiluminescence (ECL) assay kit (Amersham) according to the manufacturer's instructions.

Cyclooxygenase Activity and PGE₂ Release-- Confluent cells were made quiescent in serum-free Waymouth medium over 3 days and were then pretreated for 60 min with either 25 µM SC-58125, 25 µM ibuprofen, 20 mM sodium salicylate, 50 µM MG-132 or 18 h with 0.1 µM dexamethasone. Cells were further stimulated for the time indicated with 0.1 µM ET-1 or 50 ng/ml TNF-. When indicated, 10 µM arachidonic acid, freshly diluted in 0.1% bovine serum albumin, was added for 30 min at the end of incubation. Release of PGE₂ in the medium was assayed as described previously (6).

Assay of Protein Concentration-- Protein concentration was determined by Bio-Rad protein assay kit (Bio-Rad, France) according to the manufacturer's instructions.

Statistics-- Results are expressed as mean ± S.E. of n experiments. Statistical analysis was determined by Student's unpaired t test with p < 0.05 considered significant.

	RESULTS

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ET-1, the ETB Receptor Agonist Sarafotoxin-S6C, and TNF- Activate NF-B in Human Myofibroblastic HSC-- Nuclear proteins isolated from ET-1 or TNF--treated human myofibroblastic HSC were analyzed in electrophoretic mobility gel shift assays (EMSA), using a radiolabeled DNA probe containing the B sequence of the HIV-LTR promoter. As shown in Fig. 1A (lane 2), ET-1 increased the formation of two NF-B-DNA binding complexes. This activation was also observed with the ETB receptor agonist sarafotoxin S6C (Fig. 1A, lane 3). TNF-, which activates NF-B in numerous cell types (7-9), increased DNA binding complexes with profiles similar to ET-1 (Fig. 1A, lane 4).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Activation of NF-B by ET-1, sarafotoxin S6C, and TNF- in human myofibroblastic HSC; specificity and characterization of the NF-B-DNA complexes involved. A, EMSA of NF-B in nuclear extracts of human HSC treated with ET-1, S6C, or TNF-. Nuclear extracts were prepared from HSC made quiescent by incubation in serum-free medium over 3 days and further incubated for 60 min in the absence (lane 1) or in the presence of 0.1 µM ET-1 (lane 2), 0.1 µM S6C (lane 3), or 50 ng/ml TNF- (lane 4). EMSA was performed as described under "Experimental Procedures," using a radiolabeled probe containing the B motif of the HIV-LTR promoter. B, competition with specific and nonspecific oligonucleotides. Nuclear extracts from ET-1-treated cells were assayed for NF-B activity as described in A in the absence (lane 5) or presence of an excess of unlabeled oligonucleotide containing the B motif of the HIV-LTR promoter (lane 6), the HNF3 sequence (lane 7), or the B motif of the human COX-2 promoter (lane 8). C, characterization of the complexes activated by ET-1 and TNF-. Nuclear extracts from ET-1 (lane 9-11) or TNF- (lane 12-14)-treated HSC were incubated for 1 h with either 2 µg of control rabbit IgG (lanes 9 and 12), of p50 antibodies (19) (lanes 10 and 13), or of p65 antibody (lanes 11 and 14). EMSA was performed as described in A. Antibody supershifts, produced by binding of the p50 and p65 antibody, are identified by double arrowheads. The autoradiograms shown are representative of three experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Kinetics of NF-B activation by ET-1 and TNF- in human myofibroblastic HSC. Nuclear extracts were prepared from HSC made quiescent by incubation in serum-free medium over 3 days and further incubated for various times with 0.1 µM ET-1 (A) or 50 ng/ml TNF- (B). EMSA was performed as described under "Experimental Procedures" using a radiolabeled probe containing the B motif of the HIV-LTR promoter. The autoradiogram shown is representative of four experiments.

ET-1 and TNF- Induce the Degradation of IB- in Human Myofibroblastic HSC-- We studied IB regulation by ET-1 and TNF- in the cytoplasm of human HSC treated with ET-1 or TNF- for various times. The levels of IB- were analyzed by Western blot analysis using a specific IB- antibody. A protein with an expected size of 37 kDa was detected in cytoplasmic extracts of unstimulated cells (Fig. 3, A and B). Treatment of human HSC with ET-1 resulted in degradation of IB- after 15 min, in keeping with the time course of NF-B activation; total resynthesis was observed upon 3 h of treatment (Fig. 3A). TNF--induced degradation of IB- was observed within 2 min and was total after 5 min (Fig. 3B), in agreement with the rapid activation of NF-B by TNF- (Fig. 2B). Since resynthesis of IB- occurred when activation of NF-B was maintained, we investigated whether degradation of IB- could account for this prolonged activation of NF-B. However, no signal corresponding to the 47-kDa protein IB- was found in untreated human HSC, whereas IB- was detected in untreated Jurkat cells taken as control (Fig. 3C).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Kinetics of IB- degradation by ET-1 and TNF- in human myofibroblastic HSC. Cytoplasmic extracts were obtained from confluent HSC made quiescent by incubation in serum-free medium over 3 days and further incubated for various times with 0.1 µM ET-1 (A) or 50 ng/ml TNF- (B). Western blot measurements of IB- in the cytoplasmic extracts were performed as described under "Experimental Procedures" using a specific IB- antibody. C shows a Western blot of cytoplasmic extracts obtained from untreated HSC and Jurkat cells and was revealed with a specific IB- antibody (19).

ET-1 and TNF- Up-regulate Cyclooxygenase-2 (COX-2) via Activation of NF-B in Human Myofibroblastic HSC-- We have previously shown that the growth inhibitory effects of ET-1 are mediated by prostaglandins in human myofibroblastic HSC (6). Therefore, we next explored whether ET-1 and TNF- regulate COX-2 in these cells, through the NF-B site in the promoter of this gene (13).

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Induction of COX-2 mRNA and protein by ET-1 and TNF- in human myofibroblastic HSC. A, induction of COX-2 mRNA by ET-1 and TNF-. HSC made quiescent by incubation in serum-free medium over 3 days and further incubated for various periods with 0.1 µM ET-1 or 50 ng/ml TNF-, and total RNA was prepared. Northern blot analysis was performed using 20 µg of total RNA and hybridized with a 32P-labeled cDNA probe encoding COX-2 and G3PDH, as described under "Experimental Procedures." Typical autoradiograms of COX-2 signal are shown, which were quantified relative to G3PDH. The same results were obtained in two or three different experiments. B, Western blot analysis of COX-2 protein. Whole cell extracts obtained from quiescent HSC treated with ET-1 or TNF- for various periods. A typical autoradiogram is shown that was reproduced three times with similar results.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Effects of NF-B inhibitors on induction of COX-2 mRNA, COX-2, and COX-1 proteins by ET-1 and TNF-. HSC were made quiescent by incubation in serum-free medium over 3 days and further preincubated for 60 min with MG-132 or 30 min with sodium salicylate before treatment with 0.1 µM ET-1, 0.1 µM S6C, or 50 ng/ml TNF-. The figure shows the effect of 50 µM MG-132 (MG) and 20 mM sodium salicylate (Sal) on A, left panel, NF-B activation and IB- degradation by TNF-. EMSA was performed using nuclear extracts from quiescent HSC treated for 60 min with or without 50 ng/ml TNF- in the absence or in the presence of MG-132 or sodium salicylate. Western blot measurement of IB- was performed on the cytoplasmic extracts from the same cells, as described in Fig. 3. Right panel, COX-2 mRNA induction by ET-1 and TNF-. Total RNA was prepared from quiescent HSC treated for 3 h with ET-1 or TNF- in the absence or in the presence of MG-132 or sodium salicylate. Northern blot analysis was performed as described in Fig. 4. B, COX-2 protein induction by ET-1 and TNF-. Whole cell lysates were prepared as follows: upper part, quiescent HSC pretreated with or without 50 µM MG-132 for 60 min and further incubated with 0.1 µM S6C, ET-1, or 50 ng/ml TNF- for 8 h; lower part, quiescent HSC pretreated with or without 20 mM sodium salicylate for 30 min and further incubated with S6C, ET-1, or TNF- for 3 h. Western blot analysis was performed as described in Fig. 4, using a selective COX-2 antibody (21). C, effect of ET-1 and TNF- on COX-1 protein induction. Whole cell extracts and Western blot analysis were performed as in B, using a selective COX-1 antibody (20). In each case a typical autoradiogram is shown that is representative of results obtained in two or three experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effects of NF-B inhibitors and of COX inhibitors on cyclooxygenase activity stimulated by ET-1 and TNF-. A, effects of NF-B inhibitors on COX activity. HSC were made quiescent by incubation in serum-free medium over 3 days and further pretreated for 60 min with MG 132 (50 µM) or 30 min with sodium salicylate (20 mM) as described in Fig. 5, before treatment for 3 h with 0.1 µM ET-1 and 50 ng/ml TNF-. At the end of incubation, 10 µM of arachidonic acid was added for 30 min, and PGE2 released in the supernatants was measured as described under "Experimental Procedures." Basal PGE2 release was 120 ng/mg proteins. Results represent the mean ± S.E. of five experiments and are expressed as percent of respective control. *, p < 0.05; **, p < 0.02 versus control basal levels. B, effects of COX inhibitors on PGE2 release. Confluent quiescent cells were pretreated with either 0.1 µM dexamethasone for 18 h or for 60 min with either 25 µM SC-58125 or 25 µM ibuprofen and further incubated for 6 h in the absence or presence of 0.1 µM ET-1. PGE2 released in the supernatants was measured as described under "Experimental Procedures." Basal PGE2 release was 180 ng/mg proteins. Results represent the mean ± S.E. of three experiments and are expressed as percent of respective control. *, p < 0.05; **, p < 0.02 versus control basal levels. Inset, effect of dexamethasone on COX-2 protein expression. Western blot analysis of whole cell extracts was performed as described in Fig. 4. Results are the mean of two experiments.

The Inducible Cyclooxygenase-2 (COX-2) Mediates the Growth Inhibitory Effect of ET-1 and TNF- in Human Myofibroblastic HSC-- We have shown that ET-1 inhibits the growth of human myofibroblastic HSC (5, 6), but the effect of TNF- has not yet been investigated. As shown in Fig. 7B, TNF- dose-dependently inhibited the growth of human myofibroblastic HSC. This contrasts with the mitogenic effect of the cytokine reported in rat HSC (30). We next investigated whether induction of COX-2 is involved in the regulation of human myofibroblastic HSC proliferation. Addition of SC-58125 or dexamethasone to serum-stimulated cells strongly reduced the antiproliferative effects of ET-1 (Fig. 7A) and TNF- (Fig. 7B). These results suggest that activation of COX-2 is a key event in the pathway leading to inhibition of HSC proliferation by ET-1 and TNF-.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Dexamethasone and SC-58125 reduce the growth inhibitory effects of ET-1 and TNF-. Confluent cells were made quiescent by incubation in serum-free medium over 3 days and further pretreated for 18 h with 0.1 µM dexamethasone or for 30 min with 25 µM SC-58125, in the conditions described in Fig. 6. Cells were then stimulated with 5% human serum, together with varying concentrations of ET-1 (A) and TNF- (B), and pulsed with [3H]thymidine. [3H]Thymidine incorporation into DNA was measured as described under "Experimental Procedures." Results represent the mean ± S.E. of three experiments and are expressed as percent of respective control (control, 32,103 ± 1974 cpm; dexamethasone, 50,807 ± 3377 cpm; SC-58125, 43,876 ± 785 cpm). *, p < 0.05; **, p < 0.02 versus control.

	DISCUSSION

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We show here that ET-1 and TNF-, two factors that inhibit the growth of human myofibroblastic HSC, activate NF-B in these cells. Moreover, we demonstrate that activation of NF-B by ET-1 and TNF- results in the induction of COX-2 and leads to growth inhibition of myofibroblastic HSC. These data suggest that NF-B may play an important role in the negative regulation of human myofibroblastic HSC proliferation.

Our results constitute the first demonstration that ET-1 increases the formation of NF-B complexes that we identified as p50/p50 and p50/p65 dimers; identical complexes were also enhanced by TNF-. Stimulation of NF-B DNA binding by ET-1 involves the growth inhibitory endothelin B receptor, since this effect is reproduced by the ETB receptor agonist sarafotoxin S6C. The increase in NF-B binding correlates with the rapid and transient degradation of IB- in the cytosol of human HSC treated by ET-1 or TNF-. Since IB- is not detected in human HSC, our results suggest that degradation of IB- may initiate translocation of NF-B into the nucleus. Other stimuli that enhance NF-B in HSC are poorly characterized, but NF-B-binding activity is increased during the activation process of rat HSC triggered by oxidative stress (31). Moreover, whereas classical activators of NF-B include proinflammatory cytokines, viruses, and physical stress (7-9), stimulation by agonists that bind to G-protein-coupled receptors has barely been reported (32-36). The finding that ET-1 and TNF- activate NF-B in human myofibroblastic HSC constitutes another example of convergent pathways activated by growth inhibitory signals provided by cytokines and G-protein coupled-receptors. The signaling cascade activated upstream of IB- remains to be determined but may involve one of the recently identified IB kinases (37-40).

Among the possible target genes of NF-B in human myofibroblastic HSC, we focused on type 2 cyclooxygenase, since the promoter of this gene contains two NF-B consensus sites in its promoter region (13), and because we have demonstrated that inhibition of human HSC growth involves prostaglandin production (6). However, the presence and regulation of COX isoforms have not been previously documented in HSC. Our results demonstrate that the two COX isoforms, COX-1 and COX-2, are present in human HSC. As described in other cells, COX-1 is constitutively expressed in human HSC, but, in contrast to many other cell types, unstimulated HSC also express the COX-2 protein isoform. Activation of COX activity by ET-1 and TNF- is reduced by dexamethasone and by the selective COX-2 inhibitor SC-58125 to levels comparable to that obtained with the COX-1/COX-2 inhibitor ibuprofen. These results, together with the fact that ibuprofen, SC-58125, and dexamethasone dramatically reduce basal COX activity, indicate that, in human HSC, the major part of PGE₂ levels found in basal conditions derives from COX-2 activity. Therefore, COX-2 accounts for the constitutive COX activity. The role of the "constitutive" COX-1 isoform in human HSC remains to be explored, in particular in view of recent results showing that COX-1 may behave as a delayed response gene (41). The COX-2 isoform present in human HSC is also inducible, as shown by the rapid increase of its mRNA and protein expression in response to ET-1, sarafotoxin S6C, and TNF-. The equal potency of ET-1 and sarafotoxin S6C to activate COX-2 (Fig. 5) and the lack of effect of the ETA antagonist on ET-1-induced COX-2 expression (not shown) indicates that the ETB receptor is involved. Up-regulation of COX-2 mRNA by these factors is sustained and is associated with a long-lasting increase in protein and activity. Induction of COX-2 by ET-1 and TNF- is blunted by two different classes of NF-B blockers, the proteasome inhibitor MG-132, which blocks IB- degradation (23, 24), and the anti-inflammatory compound sodium salicylate (25), which is a potent inhibitor of NF-B, while being ineffective on purified cyclooxygenase activity (26). These NF-B inhibitors do not affect COX-1 protein expression. These results, which indicate that up-regulation of COX-2 requires NF-B activation, are consistent with the presence of an NF-B regulatory element in the promoter of the COX-2 gene (13), which is absent from that of the COX-1 gene (12). Our data are in line with recent observations implicating NF-B in COX-2 activation by interleukin-1, TNF-, or lipopolysaccharide by means of p65 antisense or NF-B inhibitors (13, 42-44).

Results from several studies indicate that COX-2 may be involved in the regulation of cell proliferation. Whereas most reports support a role of COX-2 in mitogenesis (45, 46), recent data have shown that COX-2 may also be involved in growth arrest (47, 48). Interestingly, Bornfeldt et al. (47) have recently reported that overexpression of COX-2 in human smooth muscle cells is associated with a marked inhibition of proliferation. In human HSC, blunting COX-2 activity with dexamethasone and with the selective COX-2 inhibitor SC-58125 blocks the growth inhibitory effect of ET-1 and TNF-, indicating that activation of COX-2 by either factor is a key event in the pathway leading to inhibition of HSC proliferation. We have previously shown that rapid production of prostaglandins leads to an increase in cyclic AMP and to the blockade of early events preceding human HSC proliferation such as mitogen-activated protein kinase activation (6). Our preliminary results suggest that this rapid production of prostaglandins would be ensured by stimulation of the COX-2 activity expressed in resting myofibroblastic HSC.³ In contrast, induction of COX-2 mRNA and protein by ET-1 and TNF- would result in a more sustained production of prostanoids and could be involved in the inhibition of more distal events, such as regulation of ET-1 receptors² and regulation of cell cycle components (49).

As described for COX-2, activation of NF-B has generally been linked to mitogenesis (50-54), but recent data indicate that induction of NF-B is also associated with growth arrest and cellular differentiation (14-16). In particular, NF-B stimulates the tumor suppressor gene p53 (16), and p65 interacts with the cell cycle inhibitor p21^WAF1. (15). Also, overexpression of the NF-B member c-Rel arrests the proliferation of HeLa cells by interacting with p21^WAF1 (14). Because of the cytotoxic effect of NF-B inhibitors during prolonged cell growth experiments, the present study does not provide a direct demonstration for a role of NF-B in the negative regulation of human HSC growth. Nevertheless, it is tempting to speculate that NF-B, by up-regulating COX-2 in response to ET-1 and TNF-, plays a major role in the negative regulation of human myofibroblastic HSC growth.

The physiopathological consequences of our findings in the context of liver fibrosis remains to be determined. On the one hand, the consensus sequence for NF-B is present in the promoter of several genes activated by TNF-, which are involved in the inflammatory process associated with the development of liver fibrosis. For example, monocyte chemotactic protein-1 or intercellular adhesion molecule 1 are expressed in rat HSC and up-regulated by TNF- (55, 56); however, TNF- is mitogenic for rat HSC (30). On the other hand, prostaglandins E are antifibrogenic in an in vivo model of liver fibrosis (57), and in vitro studies show that in rat HSC, prostaglandins E inhibit both collagen I production (57) and cell proliferation (58). These findings, together with the fact that ET-1 and TNF- inhibit the growth of human myofibroblastic HSC, following NF-B-dependent COX-2 induction, would be in favor of a negative regulatory role of this pathway in liver fibrogenesis.

In summary, we show here that two growth inhibitory peptides for human myofibroblastic HSC, ET-1 via its ETB receptors and TNF-, induce NF-B-dependent up-regulation of COX-2, resulting in inhibition of HSC proliferation. These results suggest that NF-B may play an important role in the negative regulation of human myofibroblastic HSC proliferation.