Pentoxifylline blocks hepatic stellate cell activation independently of phosphodiesterase inhibitory activity

Kwan S. Lee1,2, Howard B. Cottam3, Karl Houglum1, D. Bruce Wasson3, Dennis Carson3, and Mario Chojkier1

1 Department of Medicine, Veterans Affairs Medical Center, Center for Molecular Genetics; 3 The Sam and Rose Stein Institute for Research on Aging, University of California, San Diego, California 92161; and 2 Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea 135-270

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Activated, but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and smooth muscle actin (SMA) gene expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. The mechanisms leading to stellate cell activation in vivo are unknown. The characteristic hepatic oxidative stress cascade induced in rats by CCl4 markedly stimulated stellate cell entry into S phase, nuclear factor (NF)-kappa B activity, and c-myb expression. These changes were prevented by pentoxifylline, which also decreased CCl4-induced hepatic injury. As expected, cAMP-mediated phosphorylation of CREB-Ser133 was induced in vivo in stellate cells by pentoxifylline but not by its metabolite 5, an N-1 carboxypropyl derivative, which lacks phosphodiesterase inhibitory activity. Stellate cell nuclear extracts from CCl4-treated, but not from control, animals formed a complex with the critical promoter E box of the alpha -SMA gene, which was disrupted by c-myb antibodies and competed with by c-myb cognate DNA. Treatment with pentoxifylline or metabolite 5 prevented the molecular abnormalities characteristic of stellate cell activation induced by CCl4. These results suggest that induction of c-myb plays an important role in the in vivo activation of stellate cells. Pentoxifylline blocks stellate cell activation in vivo independently of its inhibitory effects on phosphodiesterases by interfering with the oxidative stress cascade and the activation of NF-kappa B and c-myb.

CREB phosphorylation; liver fibrogenesis; c-myb expression

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

OVERPRODUCTION OF COLLAGEN type I by activated hepatic stellate cells is a critical step in the development of liver cirrhosis (14-16, 26). However, the mechanisms leading to stellate cell activation in vivo remain unclear, and there is no established treatment for hepatic fibrogenesis (9).

Pentoxifylline is an alkylated xanthine, which is clinically useful for the treatment of conditions involving defective regional microcirculation (38). Although pentoxifylline, a nonspecific inhibitor of cyclic nucleotide phosphodiesterases (28), inhibits collagen gene expression in dermal fibroblasts (4), the mechanisms responsible for this effect are unknown. Moreover, the mechanisms responsible for the prevention of hepatic fibrosis by pentoxifylline in yellow phosphorus-induced hepatocellular necrosis (29) remain to be determined.

In this context, oxidative stress appears to play an essential role, through the induction of c-myb and nuclear factor (NF)-kappa B, in the activation of cultured stellate cells by collagen type I matrix or transforming growth factor (TGF)-alpha (24), suggesting that a similar mechanism may be responsible for stellate cell activation in vivo. Pentoxifylline inhibits NF-kappa B activity, resulting in a decreased expression of the human immunodeficiency virus-1 long terminal repeat (5), and prevents the lipopolysaccharide-induced production of tumor necrosis factor (TNF)-alpha (25, 32), which in turn activates NF-kappa B (18). Because NF-kappa B activity is induced by oxidative stress signals (18, 24), resulting in stellate cell activation (24), we tested whether pentoxifylline prevents the in vivo activation of stellate cells.

In this study, we analyzed the mechanisms leading to stellate cell activation in vivo in hepatic injury induced by CCl4 (3, 31). We found that oxidative stress results in enhanced NF-kappa B and c-myb binding activities, which appear to be critical in stellate cell activation, and that pentoxifylline blocks these molecular events as well as stellate cell activation. Moreover, the metabolite 5 of pentoxifylline, an N-1 carboxypropyl derivative (12, 38) that lacks phosphodiesterase inhibitory activity, also prevented the activation of stellate cells. These findings suggest that the inhibition of stellate cell activation by pentoxifylline results from blocking the oxidative stress cascade within stellate cells, rather than from its inhibition of phosphodiesterases.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. Sprague-Dawley male rats (50-60 g) each received a single intraperitoneal injection of CCl4 in mineral oil (1:3, vol/vol) at a dose of 2 ml/kg body wt (3) (CCl4, pentoxifylline, and metabolite 5 groups) or mineral oil only (control group). In addition, animals received intraperitoneal injections (100 µl) of saline (control and CCl4 groups), 200 mg/kg pentoxifylline (pentoxifylline group), or 200 mg/kg of the ester prodrug of pentoxifylline metabolite 5 (metabolite 5 group) at the following times with respect to mineral oil or CCl4 administration: -4 h, +8 h, +20 h, +32 h, and +44 h. The last injection (at +44 h) included 30 µCi of 6-[3H]thymidine (DuPont). Forty-eight hours after the CCl4 or mineral oil injection (and 4 h after they received [3H]thymidine), animals were killed and liver tissues were promptly removed, and a piece was fixed in 10% formaldehyde and embedded in paraffin for immunohistochemical staining (3). In some studies, blood samples were obtained at 24, 36, and 48 h after the CCl4 or mineral oil injection.

Cell isolation. Stellate cells were prepared from rats of the experimental groups described above by in situ perfusion and single-step density Nycodenz gradient (Accurate Chemical & Scientific, Westbury, NY), as described previously (3, 19, 24). Cells were mixed with 9.5 ml Hanks' containing 3 g/l bovine serum albumin and 8 ml of 28.7% (wt/vol) Nycodenz in sodium-free Hanks' buffer. The gradient was generated by placing 6 ml of Hanks' albumin solution on top of the liver cell mixture in a 50-ml centrifugation tube. After centrifugation (1,000 g, 4°C, 20 min), cells were aspirated from above the interface, washed twice in serum-free Dulbecco's modified Eagle's regular glucose medium, and collected. Stellate cells were identified by their typical autofluorescence at 328-nm excitation wavelength, staining of lipid droplets by oil red, and immunohistochemistry with a monoclonal antibody against desmin (3). Greater than 95% of the isolated cells were stellate cells (3, 19, 24). In some experiments, cells were cultured on a collagen type I matrix as described previously (24) and treated with pentoxifylline (100 µM) or metabolite 5 (100 µM) every day for 6 days. Cells were labeled with 2 µCi [3H]thymidine (70-80 Ci/mmol, Amersham). After 3 h of labeling, cells were harvested and [3H]thymidine incorporation into DNA was determined as described (8). Detection of alpha -SMA and cAMP-responsive element binding protein (CREB)-PSer133 in stellate cell extracts was performed by Western blot as described (8, 36) and using antibodies directed against alpha -SMA (Vector Laboratories) or CREB-PSer133 (Upstate Biotechnology).

NIH/3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum (34) and labeled with [3H]thymidine as described for stellate cells.

Nuclear extract preparation. Nuclei were prepared by a modification of the procedure described previously (7, 8, 24). Cells were homogenized in 1 ml of 5% citric acid, 0.5% Nonidet P-40, 10 mM NaF, and 10 mM Na pyrophosphate with a glass Dounce homogenizer with a loose-fitting pestle. The homogenized cells were placed above a cushion consisting of 30% sucrose and 1% citric acid. The nuclei were precipitated by a 4,000 g centrifugation at 4°C for 30 min and frozen at -70°C. DNA was isolated, extracted, and counted for [3H]thymidine incorporation as described previously (8). Gel retardation analysis of protein-DNA complexes are performed with an oligonucleotide of the putative DNA binding site, as described previously (7, 8, 24, 37). The sense oligonucleotides were NF-kappa B (5'-GGGGACTTTCCC-3') and alpha -SMA E box (5'-GATCATAAGCAGCTGAACTGCC-3'). Antibodies directed against c-myb and NF-kappa B 65 were obtained from Santa Cruz Biotechnology.

Immunohistochemistry. Liver tissue was immunostained with antisera raised against malondialdehyde-protein adducts as described previously (7, 11, 19). This antiserum is specific from malondialdehyde-lysine adducts (3, 20). A phase-contrast microscope was utilized for hematoxylin/eosin staining and immunohistochemistry with alkaline phosphatase secondary antibodies (Vector Laboratories). Cytochromes utilized were alkaline phosphatase with fast green as counterstain (Sigma Chemical). Negative control samples were processed in parallel under the same conditions, but with omission of the primary antibody.

Synthesis of pentoxifylline metabolite 5. Metabolite 5 of pentoxifylline (1-[3-carboxypropyl]-3,7-dimethylxanthine) and its ethyl ester were synthesized as described elsewhere (12). Briefly, theobromine (2 mmol) was combined with anhydrous K2CO3 (2.5 mmol) and dry dimethyl formamide (15 ml), and the mixture was brought to 75°C. The alkyl halide (2.5 mmol) was added and the mixture was stirred at 75°C for 18 h. The reaction mixture was cooled, poured into water (125 ml), and extracted with ethyl acetate (2 × 75 ml). The organic layer was dried over magnesium sulfate and evaporated to yield a white solid, which was triturated with ethyl ether. The resulting solid, analytically pure, was purified further by crystallization. H nuclear magnetic resonance spectrum, elemental analyses, and exact mass data were consistent with the assigned structure.

Superoxide assay. Human neutrophils were isolated from heparinized normal donor blood by Histopaque density gradient followed by hypotonic lysis to remove red blood cells. Cells (5 × 106) were suspended in 200 µl Hanks' balanced salt solution without phenol red and containing TNF-alpha (1 U) and pentoxifylline (100 µM) or metabolite 5 (100 µM). After 20 min, 1 ml of 120 µM cytochrome c with 100 nM N-formyl-Met-Leu-Phe (FMLP) was added. After a 10-min incubation at 37°C, the optical density (OD550) of the supernatant was determined.

Statistical analysis. Results were expressed as the mean of at least three independent experiments unless stated otherwise. The Student's t-test was used to evaluate the differences of the means between groups, with a P value of <0.05 treated as significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The role of pentoxifylline [a trisubstituted xanthine derivative (see Fig. 1) that decreases blood viscosity] on stellate cell activation in vivo was examined. Rats were treated with carbon tetrachloride, an hepatotoxin that induces liver lipid peroxidation (3) and stellate cell activation (31).


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Fig. 1.   Structure of pentoxifylline and metabolite 5. Pentoxifylline and metabolite 5 are trisubstituted xanthine derivatives. Pentoxifylline is designated chemically as 1-(5-oxohexyl)-3,7-dimethylxanthine, and metabolite 5 is an N-1 carboxypropyl derivative of pentoxifylline.

Stellate cell proliferation was assessed by the incorporation of [3H]thymidine, and activation by the expression of alpha -SMA (8, 31). The specific activity of the stellate cell DNA, an index of S phase labeling, was determined in all groups after 4 h of labeling with [3H]thymidine at 1:00 PM, to avoid potential variability related to circadian rhythm (8). The dose of [3H]thymidine was given intraperitoneally, as described previously (8). The [3H]thymidine specific activity of the stellate cell DNA increased ~10-fold 48 h after administration of CCl4 (Fig. 2A). Pentoxifylline treatment abolished the proliferation of stellate cells in animals treated with CCl4 (Fig. 2A), suggesting an inhibitory effect of pentoxifylline on stellate cell activation. As depicted in Fig. 2B, we found that hepatic stellate cells of control animals (lane 1) were activated by treatment with CCl4 (19), as determined by increased expression of alpha -SMA on Western blots of freshly isolated stellate cells (lane 2). Treatment with pentoxifylline prevented activation of stellate cells induced by CCl4 (lane 3). However, these effects of pentoxifylline could be the result of interfering with the hepatocellular injury induced by CCl4. Therefore, the degrees of hepatocellular injury and lipid peroxidation were determined in these animals.


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Fig. 2.   Pentoxifylline (PTX) and metabolite 5 (Met 5) inhibit stellate cell proliferation and activation in vivo. A: DNA specific activity, an index of S phase labeling, was determined in DNA (10 µg) purified from stellate cells isolated from at least 4 rats of each experimental group, as described in METHODS. P < 0.05 for CCl4 compared with all other groups. SE was <20% of the mean for all conditions. dpm, Disintegrations/min. B: alpha -smooth muscle actin (alpha -SMA) expression was analyzed in stellate cell lysates (10 µg of protein) isolated from control (lane 1), CCl4 (lane 2), CCl4 + pentoxifylline (lane 3), and CCl4 + metabolite 5 (lane 4) animals by Western blot as described in METHODS. alpha -SMA was detected only in CCl4 animals. Molecular markers are shown.

Although the histological degree of hepatocellular necrosis (at 48 h) was similar in the CCl4 and CCl4 + pentoxifylline groups, as indicated by liver staining with hematoxylin/eosin (not shown), pentoxifylline reduced significantly (P < 0.05) the release of alanine aminotransferase into the blood at 24, 36, and 48 h (Table 1), indicating a protective effect of pentoxifylline in this animal model of toxic hepatitis. The degree of hepatic lipid peroxidation was comparable in both groups of animals (Fig. 3). Protein adducts with malondialdehyde, a product of lipid peroxidation, were detected using specific antibodies against malondialdehyde-lysine epitopes, as reported previously (3, 19, 20). In contrast to the livers of control animals (Fig. 3A), enhanced lipid peroxidation was comparable at 48 h in zones 2 and 3 of the hepatic acini in animals treated with CCl4 alone (Fig. 3B) or with the addition of pentoxifylline (Fig. 3C).

                              
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Table 1.   Effects of pentoxifylline on serum alanine aminotransferase levels in CCl4-induced hepatotoxicity


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Fig. 3.   Induction of hepatic lipid peroxidation by CCl4. Animals were studied 48 h after mineral oil (A) or CCl4 (B and C) treatment, and liver tissue was processed for malondialdehyde (MDA)-protein adduct immunohistochemistry using specific antibodies against MDA-lysine epitopes as described in METHODS. A: no adducts were detected in mineral oil-treated animals. MDA-protein adducts are detected to a comparable degree in the cytosol of hepatocytes in zones 2 and 3 of the acinus in the livers from CCl4 (B) and CCl4 + pentoxifylline (C). Magnification ×100.

Because oxidative stress increases NF-kappa B activity (18) and NF-kappa B plays important roles in the regulation of stellate cell activation by collagen type I and TGF-alpha (24), we analyzed the potential role of NF-kappa B regulation in stellate cell activation in animals treated with CCl4. Stellate cell activation in vivo was also associated with the nuclear translocation and activation of NF-kappa B, as detected by gel shift analysis. As shown in Fig. 4A, the binding of stellate cell nuclear extracts to a NF-kappa B cognate oligonucleotide was low in quiescent cells from control animals (lane 2) but increased significantly following stellate cell activation after treatment with CCl4 (lane 3). The complex of 32P-labeled NF-kappa B oligonucleotides and nuclear extracts from activated stellate cells was competed with by a NF-kappa B cognate oligonucleotide (lane 6) but not by unrelated oligonucleotides (not shown). Of interest, pentoxifylline treatment prevented not only stellate cell proliferation and activation (Fig. 2 ) but also NF-kappa B nuclear activity (Fig. 4A, lane 4) induced by CCl4.


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Fig. 4.   Pentoxifylline and metabolite 5 block the increase in nuclear factor (NF)-kappa B and alpha -SMA binding activities of activated stellate cells. Mobility shift analysis of stellate cell nuclear extracts. Equal amounts of nuclear extracts (3 µg of DNA) were incubated with 1 ng of 32P-labeled-NF-kappa B (A) or 32P-labeled-alpha -SMA-E box (B and C) oligonucleotides. The DNA-protein complexes were resolved by electrophoresis on a 6% nondenaturing polyacrylamide gel. Position of the bound DNA is indicated by arrows. Some samples were incubated with specific antibodies, unlabeled oligonucleotide, or 1,4-dithiothreitol (DTT), as indicated by +. A: representative samples of control (lane 2), CCl4 (lane 3), CCl4 + pentoxifylline (lane 4), CCl4 + metabolite 5 (lane 5), and CCl4 + NF-kappa B oligonucleotide (lane 6). On lane 1, 32P-labeled-NF-kappa B probe was processed without nuclear extracts. B: representative samples of control (lane 2), CCl4 (lane 3), CCl4 + pentoxifylline (lane 4), CCl4 + metabolite 5 (lane 5), CCl4 + c-myb antibodies (lane 6), and CCl4 + NF-kappa B oligonucleotide (lane 7). On lane 1, 32P-labeled-alpha -SMA-E box probe was processed without nuclear extracts. C: representative examples of control (lane 2), CCl4 (lane 3), and CCl4 + DTT (lane 4). On lane 1, 32P-labeled-alpha -SMA-E box probe was processed without nuclear extracts.

Because c-myb is an important inducer of proliferation in cultured hematopoietic, smooth muscle, and stellate cells (1, 24, 33), we tested whether c-myb expression plays a role in the activation of stellate cells in vivo. As expected, the critical promoter E box of the alpha -SMA gene (6) formed complexes with nuclear extracts from freshly isolated activated stellate cells from CCl4-treated animals (Fig. 4B, lane 3), but not with nuclear extracts of freshly isolated quiescent stellate cells from control animals (lane 2). Treatment of CCl4 animals with pentoxifylline prevented the formation of a complex between the alpha -SMA-E box and stellate cell nuclear extracts (lane 4), an essential step in the activation of the alpha -SMA gene. Relevant to this study, the protein-DNA complexes were disrupted by polyclonal c-myb antibodies without the formation of a supershift (Fig. 4B, lane 6), as described for stellate cells in culture (24), but not by a NF-kappa B cognate oligonucleotide (lane 7). Preimmune serum did not affect the protein-DNA complexes (not shown). These results strongly suggest a role for c-myb (and presumably an indirect effect of NF-kappa B) on alpha -SMA gene expression, a hallmark of stellate cell activation (31).

To understand the mechanisms responsible for the blocking effects of pentoxifylline on stellate cell activation, we analyzed whether they are related to its role as a cyclic nucleotide phosphodiesterase inhibitor. A major metabolite of pentoxifylline, metabolite 5 (38), was synthesized as an ethyl ester prodrug (12) (Fig. 1) and found to lack the inhibitory activity of phosphodiesterase from human neutrophils (12).

To test the biological activities of pentoxifylline and metabolite 5 independently of hepatocellular necrosis, we studied their effects on the proliferation of primary rat stellate cells. Both pentoxifylline and metabolite 5 blocked stellate cell proliferation induced by collagen type I matrix (Fig. 5A), which is mediated by an oxidative stress cascade (24). These results indicate that pentoxifylline and metabolite 5 are able to block the oxidative stress cascade induced by collagen type I matrix (24) acting directly on stellate cells. Pentoxifylline and metabolite 5 also inhibited the proliferation of NIH/3T3 fibroblasts induced by serum with a half-maximal inhibitory concentration (~100 µM) (Fig. 5B) similar to that observed in stellate cells.


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Fig. 5.   Pentoxifylline and metabolite 5 inhibit proliferation of primary rat stellate cells. A: [3H]thymidine incorporation into DNA (2 µCi of 6-[3H]thymidine/P-60 plate for 3 h), an index of S phase, was determined in primary rat stellate cells cultured on a collagen type I matrix as described in METHODS. P < 0.05 for pentoxifylline and metabolite 5. SE was <20% of mean for all conditions. B: [3H]thymidine incorporation into DNA was determined in NIH/3T3 fibroblasts as described in METHODS. P < 0.05 for pentoxifylline (open circle ) and metabolite 5 (bullet ). SE was <30% of mean for all conditions.

Next, we determined whether pentoxifylline inhibits cAMP phosphodiesterase activity in hepatic stellate cells in vivo, leading to an increase in protein kinase A-mediated phosphorylation. This signal-transduction pathway triggers site-specific phosphorylation of the nuclear transcription factor CREB on Ser133 (17). Therefore, the induction of CREB phosphorylation at Ser133 was analyzed in nuclear extracts from freshly isolated stellate cells using an antibody against the activated, phosphorylated form of CREB (21). As depicted in Fig. 6, treatment with pentoxifylline markedly increased the expression of CREB-PSer133 (43 kDa) (lane 3), which was also detected in stellate cells from control groups in longer exposures (not shown) (21), but not in stellate cells from CCl4 (lane 2) or CCl4 + metabolite 5 (lane 4) groups. In addition to recognizing CREB, anti-CREB-PSer133 detected two other proteins. These are most likely members of the CREB-activating transcription factor (ATF) family, ATF-1 and cAMP response element modulator protein (CREM), that have phosphoacceptor sequences similar to that of CREB-PSer133 (21). Nuclear extracts of stellate cells isolated from control (Fig. 6, lane 1) and CCl4 + metabolite 5 (lane 4) animals also contain small amounts of phosphorylated ATF-1 (38 kDa) and CREM (30 kDa). Neither CREB-PSer133 nor phosphorylated members of the CREB-ATF family were detected in stellate cell nuclear extracts from CCl4-treated animals (lane 2). This study indicates that pentoxifylline, but not metabolite 5, displays cAMP phosphodiesterase inhibitory effects on hepatic stellate cells in vivo.


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Fig. 6.   Pentoxifylline, but not metabolite 5, stimulates protein kinase A-mediated phosphorylation. Phosphorylation of cAMP-responsive element binding protein (CREB) at Ser133 was analyzed in stellate cell nuclear extracts (5 µg of protein) from control (lane 1), CCl4 (lane 2), CCl4 + pentoxifylline (lane 3), and CCl4 + metabolite 5 (lane 4) animals by Western blot using antibodies against CREB-PSer133 as described in METHODS. CREB-PSer133 was detected only in CCl4 (lane 2) animals. Molecular markers are shown in kDa.

In subsequent experiments, we analyzed the effects of metabolite 5 on CCl4-induced hepatotoxicity and stellate cell activation. Treatment with metabolite 5 prevented to a considerable extent the proliferation (Fig. 2A) and activation (Fig. 2B, lane 4) of stellate cells induced by the administration of CCl4. Moreover, treatment of CCl4 animals with metabolite 5 also prevented the molecular changes characteristic of stellate cell activation, including enhanced nuclear activities of NF-kappa B and c-myb (Fig. 4). These findings suggest that the cyclic nucleotide phosphodiesterase inhibitory activity of pentoxifylline is not indispensable to block stellate cell activation or proliferation.

Coculture experiments of hepatocytes and stellate cells treated with CCl4 indicate that hepatocytes exert a paracrine stimulation of both lipid peroxidation and collagen gene expression on stellate cells (3). Because pentoxifylline and metabolite 5 interfere with the oxidative stress cascade in CCl4-induced hepatic injury (Table 1) as well as in primary rat stellate cells (Fig. 5A), we analyzed whether a similar effect on oxidative stress occurs in activated neutrophils. Both pentoxifylline and metabolite 5 prevented the production of superoxide in human neutrophils treated with TNF-alpha and FMLP (Fig. 7), known inducers of oxidative stress in these cells (2, 18, 23).


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Fig. 7.   Superoxide production in human neutrophils is prevented by pentoxifylline and metabolite 5. Induction of superoxide production by TNF-alpha and N-formyl-Met-Leu-Phe in human neutrophils (control), as described in METHODS, was prevented by pentoxifylline (100 µM) and metabolite 5 (100 µM). Values are percentage of control. P < 0.05 for pentoxifylline and metabolite 5. SE was <20% of mean for all conditions.

The redox state of the cell may alter the DNA binding affinity of activator protein-1 (AP-1) factors and c-myb (39, 40). The modulation of c-myb is probably mediated through a conserved cysteine amino acid motif (KQC43R) within the DNA binding domain (40). Therefore, we determined whether 1,4-dithiothreitol (DTT) would normalize the increased binding of stellate cell nuclear extracts from CCl4-treated animals to the alpha -SMA-E box. The addition of DTT to the nuclear extracts as described previously (40) (Fig. 4C, lane 4) normalized the increased DNA-protein complex (compare lanes 2 and 3), suggesting that the critical mechanism involves oxidation of a transcription factor, presumably the DNA binding domain of c-myb.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study, we have characterized some of the molecular mechanisms involved in the activation of stellate cells in vivo, an important step in hepatic fibrogenesis (14, 15). Moreover, we have identified a cellular pathway leading to stellate cell activation that is blocked by pentoxifylline. The inhibitory activity of pentoxifylline on cyclic nucleotide phosphodiesterases is not required to block stellate cell activation.

Our results suggest a critical role of NF-kappa B and c-myb on stellate cell activation in vivo, given that during stellate cell activation the nuclear activities of NF-kappa B and c-myb are increased and that these molecular changes and stellate cell activation are both blocked by pentoxifylline and its metabolite 5. Also, we determined that in activated stellate cells from CCl4-treated animals, c-myb contributes substantially to nuclear binding to the key E box within the promoter of the alpha -SMA gene. These findings strongly suggest that c-myb is the molecular mediator of oxidative stress on stellate cell activation and that it binds to the critical E box of the alpha -SMA gene (6), the expression of which is intrinsic to the activated phenotype of stellate cells (31).

Pentoxifylline and the metabolite 5 inhibited the enhanced NF-kappa B and c-myb activities of stellate cells in CCl4-induced hepatic injury. The dose of pentoxifylline used was not toxic to the animals, as judged by their normal behavior, lack of hepatotoxicity, and preservation of cAMP-mediated phosphorylation of stellate cells. However, pentoxifylline ameliorated the release of alanine aminotransferase, indicating a protective effect in CCl4-induced hepatotoxicity. Given that pentoxifylline inhibits the lipopolysaccharide-induced production of TNF-alpha (25, 32), it might inhibit this critical pathway in Kupffer cells, resulting in decreased leakage of alanine aminotransferase (10). However, it is improbable that the inhibitory effects of pentoxifylline and metabolite 5 on stellate cell activation/proliferation in vivo are mediated by inhibition of TNF-alpha production. TNF-alpha has no (or a small) stimulatory effect on stellate cell proliferation and no effect on stellate cell activation (9). Finally, collagen gene expression in stellate cells is inhibited by TNF-alpha , an effect incongruent with a potential role of TNF-alpha in stellate cell activation (9).

Hepatocytes can exert a paracrine stimulation of both lipid peroxidation and collagen gene expression on stellate cells (3). Potential mediators of this model of oxidative stress in stellate cells include free radicals, reactive aldehydes, or cytokines produced by hepatocytes in response to CCl4. In this context, we have demonstrated the propagation of the oxidative stress cascade to stellate cells after CCl4 treatment, as judged by activation of NF-kappa B and c-myb. The increase in this oxidative stress cascade within stellate cells suggests that this mechanism may be responsible for the molecular changes leading to stellate cell activation, as predicted from studies in cultured stellate cells (24). Several cytokines stimulate stellate cell activation, including platelet-derived growth factor (PDGF) and TGF-alpha (30). Interestingly, the induction of stellate cell activation by TGF-alpha utilizes an oxidative pathway, because it can be blocked with antioxidants (24). Similarly, PDGF requires H2O2 generation for signal transduction in vascular smooth muscle cells (35). In addition, Ras-transformed NIH/3T3 fibroblasts produced large amounts of superoxide, and their mitogenic activity was inhibited with the antioxidant N-acetyl-L-cysteine (22). Pentoxifylline also inhibited proliferation of NIH/3T3 fibroblasts. These studies support the notion that cytokine and oxidative stress pathways converge in the activation of stellate cells (24) and that pentoxifylline blocks these pathways, as suggested for superoxide production by neutrophils (13).

The binding of c-myb (and other transcription factors) to cognate DNA sequences can be modulated by the redox state of the cell (39, 40). We found that the addition of DTT, a reducing agent, normalized the increased alpha -SMA-E box binding activities of stellate cell nuclear extracts from CCl4-treated animals, suggesting an oxidative modification of a critical binding factor, such as c-myb. Future studies should assess whether this oxidative cascade involves the nuclear redox factor Ref-1 (39), which functions as a DNA repair enzyme and modulates the DNA binding activity of several transcription factors (40). Although xanthine and hypoxanthine can initiate the production of hydroxyperoxide radicals in the presence of xanthine oxidase (2, 23), they are also capable by themselves of scavenging these free radicals (2). Given that pentoxifylline and metabolite 5 are substituted xanthines (12, 38), it is likely that this structure is responsible for blocking the oxidative stress cascade in the liver of animals treated with CCl4 as well as in primary stellate cells. Indeed, we demonstrated that pentoxifylline and metabolite 5 prevent the formation of superoxide induced by TNF-alpha and FMLP, indicating that both act as scavengers of free radicals.

Increased cAMP-dependent protein kinase activity in stellate cells treated with pentoxifylline was evident by the phosphorylation of CREB on Ser133, a classic phosphoacceptor for protein kinase A (17, 21). This assay also documented the inability of metabolite 5 to affect phosphodiesterase activity in vivo. Altogether, these results indicate that the effects of pentoxifylline on stellate cell activation are independent of its phosphodiesterase inhibitory activity and are most likely related to its ability to block the propagation of oxidative stress and the induction of NF-kappa B and c-myb within the stellate cells. Studies in animals with targeted deletions of NF-kappa B or c-myb may establish the role of these genes in stellate cell activation in vivo.

Our study provides insights into the molecular mechanisms leading to hepatic stellate cell activation in vivo and the blocking effects of pentoxifylline on these pathways. The findings presented here should facilitate potential therapeutic approaches for hepatic fibrosis, a major contributor to the morbidity and mortality of patients with chronic liver diseases.

    ACKNOWLEDGEMENTS

We are grateful to M. Buck for valuable suggestions. We thank K. Pak for technical assistance and L. Masse for the preparation of this manuscript.

    FOOTNOTES

This study was supported in part by National Institutes of Health Grants GM-23200, DK-38652, DK-46971, and GM-47165 and by grants from the Department of Veterans Affairs and the American Liver Foundation. K. S. Lee was supported by a grant from Yonsei University College of Medicine (Seoul, South Korea), and K. Houglum was supported by a Clinical Investigator Award (DK-02265).

Address for reprint requests: M. Chojkier, Dept. of Medicine and Center for Molecular Genetics (9111-D), Univ. of California, San Diego, CA 92161.

Received 11 July 1997; accepted in final form 4 August 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(5):G1094-G1100