Fibronectin and cytokines increase JNK, ERK, AP-1 activity, and transin gene expression in rat hepatic stellate cells

John E. Poulos1, Jason D. Weber2, Joseph M. Bellezzo1, Adrian M. Di Bisceglie1, Robert S. Britton1, Bruce R. Bacon1, and Joseph J. Baldassare2

1 Division of Gastroenterology and Hepatology, Department of Internal Medicine and 2 Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Cytokines, growth factors, and alterations in the extracellular matrix composition may play a role in maintaining hepatic stellate cells (HSC) in the activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathways that are stimulated by these factors in HSC remain to be fully elucidated. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) family, including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), plays an important role in the cellular response to stress. The aims of this study were to investigate whether fibronectin (FN) or the inflammatory cytokines interleukin-1alpha (IL-1alpha ) and tumor necrosis factor-alpha (TNF-alpha ) activate JNK, ERK, and AP-1 activity in HSC and induce the gene expression of the matrix metalloproteinase transin. Treatment of HSC with FN resulted in an up to 4.5-fold increase in ERK activity and a 2.1-fold increase in JNK activity. IL-1alpha and TNF-alpha produced up to a fourfold increase in JNK activity and a twofold increase in ERK activity. We then compared the effects of FN, IL-1alpha , and TNF-alpha on AP-1 activity and metalloproteinase mRNA induction. All three compounds increased AP-1 binding and promoter activity, and transin mRNA levels were increased 1.8-fold by FN, 2.2-fold by IL-1alpha , and 2.8-fold by TNF-alpha . Therefore, FN and inflammatory cytokines increase MAPK activity, stimulate AP-1 activity, and increase transin gene expression in HSC. Signal transduction pathways involving the MAPK family may play an important role in the regulation of matrix metalloproteinase expression by cytokines and FN in HSC.

interleukin-1; liver; mitogen-activated protein kinase; metalloproteinase; tumor necrosis factor-alpha

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PATHOLOGICAL FIBROSIS IN THE liver is mediated by activated hepatic stellate cells (HSC), also known as lipocytes or Ito cells (13, 17). These cells have a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. Several factors have been proposed to perpetuate the fibrogenic process in activated HSC, including inflammatory cytokines and alterations in the extracellular matrix (13, 17). However, the intracellular signaling pathways that are activated by these factors in HSC are poorly understood. Recent evidence in other cell types indicates that binding to some cytokine receptors or to integrins may activate certain serine-threonine protein kinases, including extracellular signal-regulated kinase (ERK) (10, 11, 29) and c-Jun NH2-terminal kinase (JNK) (5, 26, 43). Therefore, activation of JNK and ERK might play a role in the response of HSC to hepatic injury and inflammation. JNK activation leads to the phosphorylation of c-Jun, which can then associate as c-Jun homodimers or c-Jun/c-Fos heterodimers comprising the transcription factor AP-1 (3). The activation of ERK can induce the cellular levels of c-Fos, thus also upregulating AP-1 activity (3). AP-1 influences the expression of several genes, including those for collagenase (2, 41), gelatinase (12), and transin/stromelysin (33, 38).

Alterations in hepatic matrix composition as a result of changes in the activity of metalloproteinases may play an important role in hepatic fibrogenesis (13). Activated HSC synthesize a variety of products, including collagen (14), fibronectin (FN) (34), proteoglycans (16), transin (the rat homologue of stromelysin) (42), type IV collagenase (4), and tissue inhibitors of metalloproteinases (21). Collagenases, gelatinases, and stromelysins comprise classes of matrix metalloproteinases that are important mediators of tissue remodeling in response to growth factors, cytokines, or perturbations in the extracellular matrix (24). Transin appears to be important during inflammation due to its activation of interstitial collagenases in association with plasmin (18) and its ability to degrade components of the basement membrane (24). Transin production is stimulated by a wide variety of growth factors and cytokines, and its hepatic gene expression is upregulated after partial hepatectomy (1) and carbon tetrachloride-induced injury (19). In this study, we demonstrate that FN and the two inflammatory cytokines [interleukin-1alpha (IL-1alpha ) and tumor necrosis factor-alpha (TNF-alpha )] stimulate JNK and ERK activity, increase AP-1 activity, and induce the gene expression of transin in HSC.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

HSC isolation and culture. HSC were isolated from male Sprague-Dawley rats (retired breeders; Harlan Sprague Dawley, Indianapolis, IN), utilizing a sequential pronase and collagenase perfusion technique, as we described previously (35). HSC were purified by density-gradient centrifugation using arabinogalactan, and viability and purity were assessed as previously described (35). Cells were plated on six-well uncoated plastic tissue culture dishes (Corning Glass Works, Corning, NY) for kinase assays or on T-75 flasks for isolation of RNA. Cells were grown to confluency at 37°C in an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical, St. Louis, MO) supplemented with 12 mM NaHCO3, 2 × 105 U/l penicillin, 0.2 g/l streptomycin, and 10% Cosmic calf serum (HyClone, Logan, UT). HSC were utilized for experiments between passages 5 and 7.

JNK assay. After HSC reached confluency in 6-well culture dishes, the cultures were maintained for 48 h in serum-free medium. The medium was removed, replaced with DMEM alone or DMEM containing either soluble FN (0.1-100 µg/ml) (Sigma Chemical), TNF-alpha (0.1-100 ng/ml) (Sigma Chemical), or IL-1alpha (1.5-150 ng/ml) (Genzyme, Cambridge, MA) and incubated for 15 min at 37°C. The cells were then placed on ice, rinsed twice with ice-cold phosphate-buffered saline, and lysed by incubating at 4°C for 30 min in 200 µl of solubilization buffer containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 300 mM NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 20 mM beta -glycerophosphate, 0.1 mM sodium orthovanadate, 0.01 mg/ml aprotonin, 0.01 mg/ml leupeptin, and 0.5 mM phenylmethylsulfphonyl fluoride (PMSF). The lysates were centrifuged at 2,000 g for 10 min at 4°C, and the supernatants were removed and diluted with 3 vol of the above-mentioned buffer containing 20 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, and 0.05% Triton X-100. Cell extracts (40 µg of protein) were then mixed with 10 µg of a glutathione S-transferase-c-Jun fusion protein bound to glutathione-agarose beads (26) for 18 h at 4°C. Under these incubation conditions, JNK in the cell extracts binds tightly to the c-Jun portion of the fusion protein. At the end of the incubation, the beads and associated proteins were pelleted and washed three times in a buffer containing 20 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl2, and 0.05% Triton X-100. JNK-mediated phosphorylation of the c-Jun portion of the fusion protein was performed in 30 µl of kinase buffer (pH 7.6) containing 20 mM HEPES, 20 mM MgCl2, 5 µCi of [32P]ATP (New England Nuclear, Boston, MA), 20 mM beta -glycerophosphate, 0.1 mM sodium orthovanadate, 20 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, and 20 mM ATP at 30°C for 20 min. The reactions were terminated by the addition of 2× Laemmli buffer, and the 32P-labeled c-Jun fusion protein was resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After autoradiography, quantitation of the band corresponding to the 32P-labeled c-Jun fusion protein was performed with a scanning laser densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

ERK assay. After HSC reached confluency in 6-well culture dishes, the cultures were maintained for 48 h in serum-free medium. The medium was removed, replaced with DMEM alone or DMEM containing either soluble FN (0.1-100 µg/ml), TNF-alpha (0.1-100 ng/ml), or IL-1alpha (1.5-150 ng/ml) and incubated for 15 min at 37°C. The cells were then lysed as described for the JNK assay. Anti-ERK1 antibody (2 µg) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the lysate and incubated with gentle rotation at 4°C. After 5 h, protein A-Sepharose was added and the incubation was continued overnight at 4°C. ERK1 immune complexes were washed three times with fresh solubilization buffer, then three times with LiCl buffer [0.5 M LiCl, 100 mM tris(hydroxymethyl)aminomethane (Tris) pH 7.6], and finally with assay buffer [20 mM Tris · HCl, pH 7.6, 1.5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 50 mM beta -glycerophosphate, 1 mM dithiothreitol, and 0.03% Brij 35]. The ERK1 immune complexes were pelleted by centrifugation for 5 min at 10,000 g, and the pellets were resuspended in kinase buffer (20 mM Tris · HCl, pH 7.3, 10 mM MgCl2, 50 mM ATP). For measurement of ERK activity, the immune complexes were incubated for 45 min at 30°C with myelin basic protein (10 µg per sample) and 5 µCi [32P]ATP (Amersham). The reaction was terminated by addition of 2× Laemmli buffer, and the samples were subjected to SDS-polyacrylamide electrophoresis. After autoradiography, quantitation of the band corresponding to the 32P-labeled myelin basic protein was performed with a scanning laser densitometer and ImageQuant software (Molecular Dynamics).

Electrophoretic mobility shift assay for AP-1. Confluent cultures of HSC were serum starved for 48 h and then incubated for 18 h at 37°C in DMEM with or without either FN (100 µg/ml), TNF-alpha (10 ng/ml), or IL-1alpha (15 ng/ml). Nuclear extracts were prepared using the procedure described by Schreiber et al. (37). Cells were harvested in a solution containing 20 mM Tris · HCl, pH 7.8, 5 mM MgCl2, 0.5 mM dithiothreitol, 0.3 M sucrose, 1 mM PMSF, 0.2 mM EGTA, 5 mM beta -glycerophosphate, and protease inhibitors. Nuclear extracts were obtained after centrifugation at 12,000 g, and binding assays were established in 25 µl of binding buffer, as described previously (8), using 3 µg of nuclear extract per reaction. In the binding reaction, nuclear extracts were incubated for 15 min at 20°C with 0.1 ng (10,000 counts/min) of 32P-end-labeled AP-1 consensus oligonucleotide (Santa Cruz Biotechnology). For competition studies, 50-fold excess of either unlabeled AP-1 oligonucleotide or an unrelated oligonucleotide (OCT-1) was incubated with nuclear extracts for 5 min before the addition of the labeled AP-1 oligonucleotide. To resolve the oligonucleotide-protein complex, aliquots of the binding reaction were electrophoresed on 4% nondenaturing polyacrylamide gels and after drying, radioactivity on the gels was visualized by autoradiography.

Western blotting for nuclear c-Jun and c-Fos. Confluent cultures of HSC were serum starved for 48 h and then incubated for 18 h at 37°C in DMEM with or without either FN (100 µg/ml), TNF-alpha (10 ng/ml), or IL-1alpha (15 ng/ml). Nuclear extracts were prepared as described above, and 5 µg aliquots were electrophoresed on 10% polyacrylamide gels. Separated proteins were then transferred to polyvinylidene difluoride membranes (Millipore, Boston, MA), and the membranes were probed with primary antibodies to c-Jun or c-Fos (Santa Cruz Biotechnology). Goat anti-rabbit immunoglobulin G horseradish peroxidase conjugate (Biorad) was added as the secondary antibody, and specific protein bands were visualized using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) following the manufacturer's suggested protocol.

Transfection and measurement of chloramphenicol O-acetyltransferase activity. HSC were transfected with an AP-1 responsive chloramphenicol O-acetyltransferase (CAT) reporter construct (12-O-tetradecanoylphorbol-13-acetate response element-CAT) (gift of Dr. Gary Fisher). HSC (5 × 105 cells) were plated overnight on 35-mm plates to reach 50% confluency. After a change of medium, the cells were transfected with 3 µg of purified construct DNA utilizing lipofectamine (Life Technologies, Gaithersburg, MD). The cultures were incubated for 24 h at 37°C, and the medium was replaced with fresh DMEM containing 10% Cosmic calf serum for 24 h to allow the cells to recover. The medium was then replaced with DMEM, and the cells were serum starved for 48 h before exposure to FN (100 µg/ml), IL-1alpha (15 ng/ml), or TNF-alpha (10 ng/ml) for 18 h. Cell extracts were prepared, normalized for protein, and assayed for CAT activity, as previously described (15). Quantitation of CAT activity was done by direct scanning of autoradiographs from thin-layer chromatography plates, using a scanning laser densitometer and ImageQuant software (Molecular Dynamics). The CAT assay was performed in triplicate on four independent cell preparations.

RNA isolation and ribonuclease protection assay. Untreated (control) HSC and HSC treated for 18 h with FN (100 µg/ml), IL-1alpha (15 ng/ml), or TNF-alpha (10 ng/ml) were lysed by the addition of RNA Stat-60 (1 ml/5 × 106 cells) (Teltest, Friendswood, TX). RNA was extracted with 0.2 vol of chloroform and precipitated with 0.5 vol of isopropanol. The RNA pellets were washed with 75% ethanol and resuspended in 1 mM EDTA. The ribonuclease (RNase) protection assay was performed as previously described (32) with the following modifications. Briefly, 40 µg of RNA was hybridized with an antisense [32P]UTP-labeled RNA probe prepared by in vitro transcription of a fragment isolated from the cDNA for rat transin (25). An EcoR I/BamH I fragment was isolated and subcloned into Bluescript KS plasmid (Promega, Madison, WI) with the orientation of the T7 promoter giving antisense RNA and the T3 promoter yielding sense RNA. The antisense RNA probe protected 400 bp of the transin mRNA. Sty I digest of the cDNA for glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX) (280 bp fragment) was included in the hybridization reaction as a control. Antisense riboprobes were labeled and transcribed from their respective cDNAs utilizing a transcription system (Gemini; Promega) with the addition of [32P]UTP (300 Ci/mmol). Hybridization and digestion to yield the protected fragments were performed using an RPA II kit (Ambion). Samples were electrophoresed on a 6% polyacrylamide gel, and the radioactive bands were detected by autoradiography and quantitated by scanning laser densitometry and ImageQuant software (Molecular Dynamics).

Statistical analysis. The results are expressed as means ± SE. Statistical difference between groups was assessed using two-tailed Student's t-test or analysis of variance with P <0.05 considered as significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

FN, IL-1alpha , and TNF-alpha increase JNK and ERK activity. Treatment of HSC with FN, IL-1alpha , or TNF-alpha increased JNK activity above basal levels, although unstimulated HSC showed considerable basal JNK activity (Figs. 1-3). The addition of soluble FN to HSC cultures at concentrations of 10 and 100 µg/ml produced a 1.8- and 2.1-fold increase in JNK activity and a 4.5- and 4.3-fold increase in ERK activity, respectively (Fig. 1). Treatment of HSC with FN at concentrations of 0.1 and 1 µg/ml increased JNK activity by 1.2- and 1.4-fold and ERK activity by 2.1- and 3.0-fold, respectively. IL-1alpha at concentrations of 1.5, 15, and 150 ng/ml produced significant dose-dependent increases (1.7-, 2.3-, and 4.3-fold, respectively) in the phosphorylation of the c-Jun fusion protein (Fig. 2) and a twofold increase in ERK activity (data not shown). Similar to IL-1alpha , TNF-alpha at concentrations of 1.0, 10, and 100 ng/ml elicited 2.4-, 4.7-, and 4.2-fold increases in JNK activity and a 2.2-fold increase in ERK activation (Fig. 3).


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Fig. 1.   Fibronectin (FN) stimulates c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in hepatic stellate cells (HSC). HSC were incubated in absence or presence of FN (0.1-100 µg/ml) for 15 min. JNK and ERK activity were measured in cell extracts by determining phosphorylation of a c-Jun fusion protein (top) and myelin basic protein, respectively (see METHODS). Results are expressed as fold increase over control values (means ± SE; n = 5).


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Fig. 2.   Interleukin-1alpha (IL-1alpha ) stimulates JNK in HSC. HSC were treated with IL-1alpha (1.5, 15, or 150 ng/ml) for 15 min. IL-1alpha caused progressive increase in JNK activity, indicated by increased phosphorylation of c-Jun fusion protein. Results are expressed as fold increase over control values (means ± SE; n = 5).


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Fig. 3.   Tumor necrosis factor-alpha (TNF-alpha ) increases JNK and ERK activity in HSC. HSC were incubated with TNF-alpha (0.1-100 ng/ml) for 15 min. JNK and ERK activity were measured by determining phosphorylation of c-Jun fusion protein (top) or myelin basic protein, respectively. Results are expressed as fold increase over control values (means ± SE; n = 3).

FN, IL-1alpha , and TNF-alpha increase AP-1 binding activity. To determine if FN, IL-1alpha , and TNF-alpha stimulate AP-1 in HSC, an electrophoretic mobility shift assay was performed to measure AP-1 binding activity in nuclear extracts. Figure 4 shows that FN, IL-1alpha , and TNF-alpha increased nuclear AP-1 binding in HSC. The shifted band was competitively inhibited by the addition of a 50-fold excess of unlabeled AP-1 nucleotide but not by an excess of an unrelated oligonucleotide (OCT-1).


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Fig. 4.   FN, IL-1alpha , and TNF-alpha increase AP-1 nuclear binding activity in HSC. HSC were serum starved for 48 h and incubated with FN (100 µg/ml), TNF-alpha (10 ng/ml), or IL-1alpha (15 ng/ml) for 18 h. Nuclear extracts were subsequently incubated in presence of radiolabeled AP-1 oligonucleotide with or without 50-fold excess of either unlabeled AP-1 (50× cold AP-1) or an unrelated oligonucleotide (50× cold OCT-1). FN, IL-1alpha , and TNF-alpha increased AP-1 binding activity, relative to that seen in untreated control cells (CTL). Shifted band induced by agonist treatment was inhibited by excess unlabeled AP-1 oligonucleotide but not by excess OCT-1 oligonucleotide. Results are representative of 3 independent experiments.

Western blotting for nuclear c-Jun and c-Fos. To determine the nuclear levels of c-Jun and c-Fos, HSC were treated with FN (100 µg/ml), IL-1alpha (15 ng/ml), or TNF-alpha (10 ng/ml) for 18 h and nuclear extracts were prepared and examined by Western analysis. As shown in Fig. 5, IL-1alpha , TNF-alpha , and FN caused an approximately twofold increase in nuclear c-Jun levels. IL-1alpha and TNF-alpha also increased the nuclear levels of c-Fos by about twofold. Unstimulated HSC contained detectable basal levels of c-Jun and c-Fos. The increases in the nuclear levels of the AP-1 family members, c-Jun and c-Fos, coincided with elevations in AP-1 binding and AP-1 dependent gene expression (Figs. 4 and 6).


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Fig. 5.   Effect of FN, IL-1alpha , and TNF-alpha on nuclear levels of c-Jun and c-Fos. Cells were serum starved for 48 h and incubated with FN (100 µg/ml), IL-1alpha (15 ng/ml), or TNF-alpha (10 ng/ml) for 18 h. Levels of c-Jun and c-Fos proteins were analyzed by Western blotting of nuclear extracts from control (C) HSC and HSC treated with TNF-alpha (T), IL-1alpha (IL), or FN. Results are representative of 3 independent experiments.


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Fig. 6.   FN, IL-1alpha , and TNF-alpha increase expression of AP-1 responsive reporter construct in HSC. HSC were transfected with an AP-1 responsive 12-O-tetradecanoylphorbol-13-acetate response element (TRE)-chloramphenicol O-acetyltransferase (CAT) construct (see METHODS). Transfected cells were serum starved for 48 h and then exposed to FN (100 µg/ml), IL-1alpha (15 ng/ml), or TNF-alpha (10 ng/ml) for 18 h. After cells were harvested, cellular CAT activity was measured. Two bands represent diacetylated (top) and monoacetylated (bottom) chloramphenicol. Results are expressed as fold increase over control values (means ± SE; n = 4). * P < 0.05.

FN, IL-1alpha , and TNF-alpha increase expression of an AP-1 responsive reporter construct. Transient transfections of HSC with an AP-1 responsive reporter construct were performed to determine if the observed elevations in AP-1 binding activity were associated with increased transcription of an AP-1 responsive CAT construct. FN, IL-1alpha , and TNF-alpha elicited a 1.5-, 2.8-, and 2-fold increase, respectively, in CAT activity relative to the activity in unstimulated cells (Fig. 6).

FN, IL-1alpha , and TNF-alpha increase transin mRNA levels. An RNase protection assay was used to measure the levels of transin mRNA in HSC (Fig. 7). Treatment with FN resulted in a 1.8-fold increase (P < 0.01) in transin mRNA levels, whereas treatment with IL-1alpha and TNF-alpha caused a 2.2- and 2.8-fold increase, respectively (P < 0.001). These data demonstrate that FN, IL-1alpha , and TNF-alpha increase the steady-state levels of transin mRNA in HSC.


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Fig. 7.   FN, IL-1alpha , and TNF-alpha increase transin mRNA levels in HSC. RNA was isolated from untreated control cells and from cells treated for 18 h with either FN (100 µg/ml), IL-1alpha (15 ng/ml), or TNF-alpha (10 ng/ml). mRNA levels for rat transin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured using antisense riboprobes in an RNase protection assay. After autoradiography, protected bands were quantitated using scanning laser densitometry and values were calculated as ratio of transin band to GAPDH band. Results represent fold increase over control values (means ± SE; n = 4). * P < 0.005.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Activated HSC are the major source of extracellular matrix components during hepatic fibrosis (13, 17). Activated HSC have been observed in human liver disease and in a variety of animal models of hepatic fibrosis, including iron overload, chronic cholestasis, ethanol administration, and chronic carbon tetrachloride (7, 13, 17). The perpetuation of the activated phenotype of HSC may depend on a complex interplay between inflammatory cytokines, altered extracellular matrix, and continued hepatocellular injury (13, 17). In the present study, we demonstrate that FN and two inflammatory cytokines (IL-1alpha and TNF-alpha ) activate JNK, ERK, and AP-1 in HSC and upregulate transin gene expression in these cells. The activation of these mitogen-activated protein kinase (MAPK) signaling pathways by FN, IL-1alpha , and TNF-alpha may contribute to their effects on the fibrogenic state of HSC.

The deposition of FN is increased early in inflammation (30), and FN is a constituent of the abnormal matrix laid down in hepatic fibrosis (22). FN is found in two main forms: a circulating form and a cellular form (30). Circulating levels of FN have been reported to be ~300 µg/ml (28), and the concentration of cellular FN present within the extracellular matrix of the liver may vary according to the degree of injury and inflammation. Both forms of FN contain an RGD cell-binding sequence that can bind to the alpha 5beta 1-integrin in cell plasma membranes, and this integrin has recently been detected in cultured human HSC (9). In other cells, activation of MAPK by FN appears dependent on integrin binding, receptor aggregation, and tyrosine kinase activity (27). Recent evidence indicates that, unlike circulating FN, the cellular FN produced during hepatic injury contains a segment (EIIIA) that is particularly efficacious in activating HSC (22). Because the current experiments used the circulating form of FN to activate ERK and JNK, it is not known what effect hepatic cellular FN would have on these signaling pathways.

Previous studies in rabbit fibroblasts indicate that the effects of FN on the expression of transin/stromelysin may be complex. When these fibroblasts were plated on dishes coated with FN, the expression of stromelysin was not changed, but when a mixed substrate of FN and tenascin was used, it caused induction (39). In addition, plating these cells on a fragment of FN containing the RGD region caused induction of stromelysin, whereas another region of the molecule inhibited stromelysin expression through binding to the alpha 4beta 1-integrin (20). It is not known whether HSC express the alpha 4beta 1-integrin. Because our experiments used HSC attached to plastic and endogenously produced matrix, the effects of soluble FN that we observed may have been influenced by cooperative effects of attachment to other extracellular matrix molecules. Characterization of the integrin composition of rat HSC and the effects of attachment to different matrices will be useful to more fully evaluate possible integrin-mediated signaling in HSC.

Hepatic inflammation elicits the release of a wide variety of cytokines, including IL-1alpha and TNF-alpha , from activated macrophages and other inflammatory cells. The ability of IL-1alpha and TNF-alpha to stimulate JNK in HSC is consistent with previous reports in fibroblasts (5) and monocytes (43). The four- to fivefold increase in JNK activity induced by IL-1alpha and TNF-alpha in HSC is lower than the increases reported in some other cell types (5, 43). Besides the possibility of an inherent difference in responsiveness of HSC to these agonists, the high basal JNK activity in unstimulated HSC may have decreased the fold stimulation seen with IL-1alpha and TNF-alpha .

The current results indicate that FN, IL-1alpha , and TNF-alpha stimulate both JNK and ERK in HSC, with IL-1alpha and TNF-alpha having a more pronounced effect on JNK activity, whereas FN is a more potent stimulant of ERK than JNK. Evidence is accumulating from a variety of cell types that several mediators can activate multiple MAPK pathways, albeit with different efficacies (11). One potential site of integration of JNK and ERK signaling is at the level of the transcription factor AP-1 (3). A major target of JNK is the transcriptional activation domain of c-Jun: c-Jun forms the AP-1 transcription factor either as a homodimer or as a heterodimer with c-Fos (3, 23). The activation of ERK can induce the cellular levels of c-Fos thus also stimulating AP-1 activity (3, 23). AP-1 regulates transcription at promoters containing the 12-O-tetradecanoylphorbol-13-acetate-response element, and previous work in several cell types has indicated a role for AP-1 in regulating transin/stromelysin gene expression (33, 38). The present study demonstrates that FN, IL-1alpha , and TNF-alpha activate both AP-1 activity and transin mRNA levels in HSC. Alcorn et al. (1) have observed that the hepatic mRNA levels of c-Jun and transin are increased after partial hepatectomy, and they suggested that c-Jun-dependent AP-1 activity may be responsible for the induction of transin gene expression.

This study shows that FN, IL-1alpha , and TNF-alpha increase nuclear AP-1 binding activity and the expression of an AP-1 reporter construct in HSC. The induction of AP-1 binding activity (as determined by electrophoretic mobility shift assay) was greater than the increase in AP-1 dependent gene expression (as determined in transfected HSC). This is not unexpected, since changes in AP-1 DNA binding do not always reflect the transcriptional activity of this complex (23). The ability of AP-1 to activate transcription is best assessed using an AP-1 reporter gene in transfection experiments (23), and the current results indicate that IL-1alpha , TNF-alpha , and FN elicit a 2.8-, 2.0-, and 1.8-fold increase, respectively, in the expression of an AP-1 CAT reporter construct in HSC. This correlates well with the increases in steady-state transin mRNA levels produced by these three substances.

The effects of IL-1alpha and TNF-alpha on mRNA levels of AP-1 family members and the constituents of the AP-1 complex have been examined in other cell types. Treatment with these agonists elicits early increases in c-Fos and c-Jun mRNA levels (6, 31, 41). The cell attachment region of FN has also been shown to elicit elevations in c-Fos and c-Jun mRNA that are associated with the subsequent expression of collagenase (40). Thus the current consensus is that early increases in c-Jun and c-Fos gene transcription lead to increases in the levels of these proteins, with the formation of heterodimers of c-Fos and c-Jun or homodimers of c-Jun, which can bind to AP-1 sites in DNA. In addition, the activation of JNK results in the phosphorylation of c-Jun, which increases its transcriptional activity, and this mechanism may be responsible for part of the increase in AP-1 activity (23). We have determined by Western blotting that TNF-alpha , IL-1alpha , and FN elicit increases in nuclear c-Jun levels, whereas TNF-alpha and IL-1alpha also increase nuclear c-Fos, in association with elevations in AP-1 activity in HSC. In accordance with what has been observed in other cell types, it seems most likely that c-Jun homodimers and c-Jun/c-Fos heterodimers are the major constituents of the AP-1 complex that is induced by TNF-alpha , IL-1alpha , and FN in HSC. However, these results do not exclude the possibility of a role for other AP-1 family members in FN- and cytokine-mediated transcriptional activation within HSC.

In conclusion, the results of the current experiments suggest that activation of the JNK and ERK pathways by FN and inflammatory cytokines leads to an increase in AP-1 activity and transin gene expression in HSC. The upregulation of the gene expression for transin might result in increased production of this metalloproteinase, which could have several possible consequences, including activation of extracellular proteases in their proenzyme form and degradation or alteration of ECM components, which may further perpetuate HSC activation. Further understanding of terminal events elicited through the MAPK signaling pathways in HSC may provide insight into the mechanisms by which some inflammatory cytokines and FN may influence the fibrogenic activity of these cells.

    ACKNOWLEDGEMENTS

This study was supported by a National Research Service Award F-32 (to J. E. Poulos), National Institutes of Health Grants RO1 DK-41816 (to B. R. Bacon) and RO1 HL-40901 (to J. J. Baldassare), a Howard Hughes Medical Institute Medical Student Research Training Fellowship (to J. M. Bellezzo), and by a donation from the David Lichtenstein Foundation (to B. R. Bacon).

    FOOTNOTES

Portions of this study were presented at the annual meetings of the American Association for the Study of Liver Diseases (Chicago, IL, November 1995, 1996) and have been published in abstract form (Hepatology 22: 293A, 1995; Hepatology 24: 459A, 1996).

Address for reprint requests to: B. R. Bacon, Division of Gastroenterology and Hepatology, St. Louis Univ. Health Sciences Center, 3635 Vista Ave., St. Louis, MO 63110-0250.

Received 13 August 1996; accepted in final form 4 June 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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