TNF-alpha inhibits liver collagen-alpha 1(I) gene expression through a tissue-specific regulatory region

Karl Houglum, Martina Buck, Dong Joon Kim, and Mario Chojkier

Department of Medicine, Veterans Affairs Medical Center, and Center for Molecular Genetics, University of California, San Diego, California 92161

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Although tumor necrosis factor-alpha (TNF-alpha ) inhibits collagen-alpha 1(I) gene expression in cultured hepatic stellate cells, assessment of its effects on hepatic collagen expression is complicated by the confounding variables of tissue necrosis and inflammation. Therefore, we analyzed whether chronically elevated serum TNF-alpha affects constitutive hepatic collagen metabolism in vivo by inoculating nude mice with Chinese hamster ovary (CHO) cells secreting TNF-alpha (TNF-alpha mice) or with control CHO cells (control mice). Before the onset of weight loss, collagen synthesis and collagen gene expression were inhibited in the liver of TNF-alpha mice. In transgenic mice, after 8 h, TNF-alpha (500 ng at 0 and 5 h) inhibited the liver expression of the collagen-alpha 1(I)-human growth hormone (hGH) transgene containing the first intron and -440 bp of the 5' region. Similarly, in cultured hepatic stellate cells isolated from these transgenic animals, the -440 bp collagen-alpha 1(I)-hGH transgene was responsive to TNF-alpha treatment independent of the activation of these cells. Transfection studies in stellate cells allowed further characterization of this TNF-alpha -responsive segment to -220 bp of the 5' region. Because in the skin the inhibitory effect of TNF-alpha involves a regulatory region of the collagen-alpha 1(I) gene beyond -440 bp, we herein identify a novel tissue-specific regulation of collagen-alpha 1(I) gene by TNF-alpha .

liver fibrosis; cytokines; cachexia; transcription; stellate cells

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

TUMOR NECROSIS FACTOR-alpha (TNF-alpha ) is secreted by macrophages in response to inflammation, infection, and cancer (4). TNF-alpha plays a major part in tissue inflammation (53) and remodeling by stimulating production of collagenase gene expression (6). Also, TNF-alpha inhibits collagen-alpha 1(I) gene expression in cultured fibroblasts (50) and in activated hepatic stellate cells (lipocytes or Ito cells) (2). In contrast, TNF-alpha has been implicated as a profibrotic agent in lung inflammation and fibrosis induced by silica or bleomycin (41, 42), and expression of a TNF-alpha transgene in murine lung causes fibrosing alveolitis (38). These data suggest that the effects of TNF-alpha on fibrogenesis may be different in various pathological conditions, such as those associated with lung inflammation (41, 42).

Although elevated serum TNF-alpha has been found in chronic diseases of the liver and other tissues (9, 22, 36, 48, 52), its role in hepatic fibrosis, particularly the effects on hepatic collagen gene expression, remains unknown. The assessment of this important issue is complicated by the confounding variables of tissue necrosis and inflammation associated with tissue injury. These confounding variables could be avoided by analysis of constitutive collagen gene expression. Therefore, we analyzed the role of TNF-alpha on hepatic stellate cell collagen gene expression independent of hepatic necroinflammation or stellate cell activation.

We found that both chronic and short-term administration of TNF-alpha inhibit constitutive liver collagen-alpha 1(I) gene expression. In addition, we established that in the liver or in cultured hepatic stellate cells isolated from transgenic mice, the -440 bp 5' segment and the first intron of the collagen-alpha 1(I) gene were sufficient for responsiveness to TNF-alpha treatment. Furthermore, transfection of chimeric reporter genes to hepatic stellate cells allowed us to circumscribe the TNF-alpha responsiveness to the -220 bp 5' region. Because in the skin the inhibitory effect of TNF-alpha involves a regulatory region of the collagen-alpha 1(I) gene beyond -440 bp (8), we herein identify a novel tissue-specific regulation of collagen-alpha 1(I) gene by TNF-alpha .

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Mouse model of cachexia. Chinese hamster ovary (CHO) cells were stably transfected with either the human TNF-alpha gene cloned into a mammalian expression vector (TNF-alpha cells) or the mammalian expression vector alone (CHO cells, control) as described previously (5, 7, 8). Cells were grown in DMEM supplemented with 10% FCS, penicillin (50 U/ml), and streptomycin (50 µg/ml). The TNF-alpha cells, but not the CHO cells, produced TNF-alpha as measured by either cytolytic or ELISA assays. Four-week-old male nude mice were injected intramuscularly with either 107 CHO cells or 107 TNF-alpha cells as described previously (5, 7, 8). Nude mice were housed in a temperature- and humidity- controlled facility. Animals had free access to food and water, and they were killed at 14-21 days (before the onset of weight loss of the TNF-alpha mice). Serum TNF-alpha levels were determined by an immunoassay using monoclonal antibodies against human TNF-alpha (Endogen) (5, 7). In other experiments, adult C57BL/6 mice were injected intraperitoneally with either 500 ng of human recombinant TNF-alpha or saline (control) at 0 and 5 h and killed at 8 h. At least three animals were used for each group in all experiments.

Determination of specific activities of liver collagen and noncollagen proteins. Mice were fed ad libitum and had free access to water. Animals were given intraperitoneal injections of 200 µCi of [5-3H]proline in 0.5 ml of sterile saline, and after 2 h they were killed and the liver was removed and placed immediately in ice-cold buffer A (10, 12). This buffer contains 50 mM Tris · HCl, 15 mM EDTA, 1 M NaCl, 1 mM phenylmethylsulfonyl fluoride, and 5 mM N-ethylmaleimide, pH 7.5. The liver was homogenized for 1 min at speed setting 7 of a DuPont Sorvall Omni-Mixer. Portions of liver homogenates were used to determine the specific activity of liver proteins, as described below. The remainder was used to purify collagen as described previously (10). After centrifugation of liver homogenates at 3,000 g for 30 min at 4°C, the precipitates were suspended in 0.5 N acetic acid containing pepsin (10 mg/g) and were incubated for 6 h at 4°C with stirring. The suspensions were centrifuged at 3,000 g for 30 min at 4°C; the supernatant solutions were adjusted to pH 7.5 with 2 N NaOH. The pepsin digestion was repeated twice. The supernatants were combined and precipitated with 176 mg/ml (NH4)2SO4, pH 8, and the precipitates were collected by centrifugation at 14,000 g at 4°C for 1 h. A second (NH4)2SO4 precipitation of pepsin-purified collagen was carried out and the precipitates were washed with 10 ml 70% ethanol and dissolved in 0.1 N NaOH. The amount of collagen was determined by the absorbance at 562 nm produced with the bicinchoninic acid (BCA) colorimetric assay (sample:reagent, 1:20; 50°C for 40 min) using calf skin collagen as a standard. Portions of the NaOH solutions were also used to measure the radioactivity. The liver collagen specific activity was calculated from the radioactivity incorporated into collagen and expressed as dpm/mg of collagen.

Liver proteins were precipitated from liver homogenates three times with 66% ethanol, and the precipitate was collected at 3,000 g at 4°C for 10 min. The precipitate was then solubilized with 0.2 N NaOH and stored at -20°C. The specific activity of the total liver proteins (dpm/mg) was determined by the amount of protein measured with the BCA colorimetric assay, using albumin as standard, and the radioactivity quantified by liquid scintillation spectroscopy.

SDS-PAGE. SDS-PAGE was performed as described previously (10). An LKB vertical slab gel was employed with SDS buffer on a discontinuous system. Some samples were reduced with 100 mM dithiothreitol at 95°C for 2 min. Electrophoresis was carried out at 45 mA/gel for 2 h. Identification of bands was done with either 0.25% Coomassie blue dye in 50% methanol or 10% acetic acid using protein standards.

Determination of collagen-alpha 1(I) mRNA. RNA was isolated from liver and quantitated by a sensitive RNase protection assay as described previously (6-8, 18, 26), using the riboprobes for 18S RNA (Ambion), exon 5 of the human growth hormone (hGH) gene, mouse collagen-alpha 1(I), and beta 2-microglobulin (26, 39, 49). Collagen-alpha 1(I) mRNA and hGH mRNA values were measured simultaneously with the internal standards beta 2-microglobulin mRNA and 18S RNA, respectively, as described by us previously (6-8, 18, 26).

Expression of chimeric collagen-alpha 1(I) hGH reporter genes in the liver. Transgenic mouse lines expressing hGH transgenes were as described by Bornstein and co-workers (34, 49). We used a transgenic line containing portions of the human alpha 1(I) collagen gene, -2300 COL (bases -2300 to +1607) and -440 COL (bases -440 to +1607), which include the first intron and first exon. The -440 COL line has been previously described (26, 49) and contains two copies of the transgene, whereas the -2300 COL line contains 10-12 copies (26). The cis-regulatory region of the collagen-alpha 1(I) gene responsive to TNF-alpha was characterized in transgenic mice expressing hGH under the direction of -2,300 bp or -440 bp of the human collagen-alpha 1(I) with the first intron (49). At least three animals were used for each group in all experiments.

Cell cultures. Primary hepatic stellate cells from transgenic mice were isolated essentially as described previously (26). Briefly, after the livers were excised and washed in Hanks' balanced salt solution (HBSS) they were minced and incubated at 37°C for 30 min with constant shaking with 0.5% Pronase (Boehringer-Mannheim), 0.05% collagenase B (Boehringer-Mannheim), and 10 µg/ml DNase (United States Biochemical) in HBSS without Ca2+. This digest was filtered through gauze and pelleted at 450 g for 10 min and subsequently rinsed four times with HBSS containing 10 µg/ml DNase. Stellate cells were purified by a single-step density Nycodenz gradient (Accurate Chemical and Scientific, Westbury, NY), as described previously (3, 26, 32). Cells were cultured on EHS (Matrigel) basement membrane matrix (Becton-Dickinson) and used after 6 days of culture. Stellate cells growing on an EHS matrix were activated with FeSO4 (50 µM)/ascorbate (200 µM), which induces oxidative stress, as we have described previously (32). Cells were cultured under an atmosphere of 5% CO2-95% air, in DMEM containing 10% FCS. 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). In addition, the activation of stellate cells was determined by the expression of alpha -smooth muscle actin (alpha -SMA), judging by the immunofluorescence using specific antibodies against alpha -SMA (Sigma) (32).

Day 10-activated hepatic stellate cells were transfected with either pCol2LUC or pCol3LUC, which contain the -2300/+110 bp or the -220/+110 bp 5' region of the mouse collagen-alpha 1(I) gene (25), respectively, inserted into pGL3-Basic (Promega, Madison, WI), or pGL3-Basic alone. The efficiency of the transfection was determined in control and TNF-alpha -treated cells, by transfecting pLUC (pGL3 Basic) as described previously (27). To increase the transfectibility of activated stellate cells, a transfection-enhancing reagent (Life Technologies, Gaithersburg, MD) was added in conjunction with lipofectamine as recommended by the manufacturer. Cells were treated for 48 h with 10 ng/ml rhTNF-alpha (R & D Systems, Minneapolis, MN).

Statistical analysis. All results are expressed as means ± SE. Student's t-test was used to evaluate the differences of the means between groups, accepting P < 0.05 as significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Two weeks after their inoculation with TNF-alpha cells, mice had serum TNF-alpha concentrations of 275 ± 75 pg/ml as measured by a specific immunoassay, comparable to those reported previously (5, 7, 8). These serum TNF-alpha concentrations are lower than those utilized in experiments with cultured fibroblasts and hepatic stellate cells (2, 50). More importantly, similar or higher TNF-alpha serum levels have been found in patients with infectious, parasitic, and neoplastic diseases (22, 31, 48, 54). During the study period (3 wk) the weights of the CHO and TNF-alpha mice were comparable to those of normal nude mice as reported previously (5, 7, 8). Collagen synthesis in the liver was measured in vivo in TNF-alpha mice before they began to lose weight. After the intraperitoneal administration of [3H]proline, the specific activities of collagen and noncollagen proteins were determined. This sensitive assay allows an accurate measurement of collagen synthesis because it corrects for changes in the specific activity of the amino acid precursor pool and it is not affected by variable recoveries of proteins (8, 10, 12). The incorporation of radioactivity into liver proteins was found to be linear for up to 6 h as described previously (10, 12). During this period, the specific activity of newly synthesized liver collagen (relative to the specific activity of noncollagen liver protein) was significantly decreased (~50%) in the TNF-alpha mice compared with CHO mice (Table 1). The specific activity of total noncollagen proteins in the liver was not affected in the precachectic TNF-alpha mice (Table 1), in agreement with studies in cultured fibroblasts (50). However, TNF-alpha also inhibits albumin gene expression in precachectic mice (5). These findings indicate that TNF-alpha inhibits collagen type I production independent of factors related to weight loss (12, 13, 40). The purity of the extracted liver collagen in TNF-alpha and CHO mice was demonstrated by comparison with collagen standards on PAGE (Fig. 1A, lanes 1 and 2). In addition, the purity of the extracted liver collagen was confirmed by its susceptibility to digestion with highly specific collagenase (13, 50) (Fig. 1A, lane 3). The yield of collagen, although variable, was comparable in CHO and TNF-alpha mice. In similar purification procedures, we have demonstrated that hepatic collagen had no contaminant proteins as determined by the ratio of hydroxyproline to proline and hydroxyproline (10).

                              
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Table 1.   Analysis of collagen production in CHO and TNF-alpha mice


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Fig. 1.   Tumor necrosis factor-alpha (TNF-alpha ) inhibits liver collagen production and collagen-alpha 1(I) mRNA. Liver collagen mRNA was analyzed 3 wk after CHO or TNF-alpha cell inoculation. At least 3 animals were used for each group in all experiments. A: equal amounts of purified liver collagen from CHO (lanes 1 and 3) and TNF-alpha (lane 2) animals were applied to SDS-PAGE. Cross-linked collagen (beta  and gamma ) is apparent in lanes 1 and 2. Lane 3 was treated with purified bacterial collagenase. Migration of standard collagen markers was as indicated. B: analysis of liver collagen-alpha 1(I) gene expression in TNF-alpha mice. Total RNA was purified from liver, and collagen-alpha 1(I) and beta 2-microglobulin mRNA were measured by an RNase protection assay. Collagen-alpha 1(I) mRNA values were corrected by internal standard beta 2-microglobulin mRNA; P < 0.05 for TNF-alpha mice. C: representative examples of collagen-alpha 1(I) (110 nucleotides) protected band for CHO mice (lanes 1 and 3), and TNF-alpha mice (lanes 2 and 4) are shown.

To determine whether the decreased collagen synthesis in the liver of precachectic TNF-alpha mice was the result of an inhibition of collagen-alpha 1(I) gene expression, we analyzed collagen mRNA levels. Total RNA was purified from the liver of TNF-alpha and CHO mice, and the abundance of collagen-alpha 1(I) mRNA was measured after solution hybridization with a specific probe by a sensitive RNase protection assay (6-8, 18, 26). As expected, the protected segment of the collagen-alpha 1(I) mRNA was 110 bases, confirming the specificity of the assay. The levels of collagen-alpha 1(I) mRNA (corrected by the steady-state levels of beta 2-microglobulin mRNA) were decreased in the liver of TNF-alpha mice (Fig. 1, B and C), indicating a selective effect of chronically elevated TNF-alpha on collagen-alpha 1(I) gene expression in the liver, similar to that found in cultured fibroblasts and stellate cells (2, 50).

To characterize the TNF-alpha -responsive region within the collagen-alpha 1(I) gene, we performed experiments with transgenic mice expressing the hGH reporter under the direction of regulatory regions of the human collagen-alpha 1(I) gene (49). We have reported kinetic studies, indicating that in 20- to 25-g mice the intraperitoneal injection of 500 ng of TNF-alpha results in a peak TNF-alpha serum level of ~20 ng/ml at 45 min, decreasing to ~4 ng/ml at 3 h (1). These TNF-alpha values are comparable to those found in cachectic TNF-alpha mice (5, 7, 8) and in cachectic patients (22, 48, 54). Both the hGH transgene mRNA and the endogenous collagen-alpha 1(I) mRNA were detected by a sensitive and specific RNase protection assay, and measurement of 18S RNA was used as an internal standard (34, 49). In addition, the confounding variable of a different transgene copy number when the two lines were compared (49) was avoided by assessing expression under baseline conditions and after TNF-alpha treatment in the same transgenic line (see Figs. 2 and 3). TNF-alpha inhibits collagen-alpha 1(I) gene transcription in cultured human and murine fibroblasts during a 4- to 8-h incubation (50). Similarly, the expression of the -2300 COL-hGH transgene or the -440 COL-hGH transgene (Fig. 2A) was decreased in the liver in parallel to the endogenous collagen gene, 8 h after treatment with human recombinant TNF-alpha (500 ng at 0 and 5 h) (Fig. 2, B and C). The apparent decreased baseline expression of the hGH in the liver of the -440 COL-hGH transgene compared with the -2300 COL-hGH transgene (~8-fold), reflects a five- to sixfold difference in copy number, as well as a small difference in riboprobe specific activity (26, 34) (Fig. 2C).


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Fig. 2.   TNF-alpha inhibits liver expression of human growth hormone (hGH) in transgenic animals bearing -2300 or -440 COL-hGH minigenes. A: schematic representation of human collagen-alpha 1(I)-hGH minigenes. These constructs were used for the generation of transgenic mice. The 5' flanking regions are shown as open rectangles. Sequences of the first exon and first intron of collagen-alpha 1(I) genes are indicated in solid and hatched rectangles, respectively. Arrows denote start of transcription. Reporter gene sequences of hGH (exons 4 and 5) are indicated in gray rectangles. B: expression of collagen-alpha 1(I)-hGH minigenes. Transgenic mice received intraperitoneal injections of saline (control) or TNF-alpha as described in METHODS, and collagen-alpha 1(I) mRNA was analyzed by RNase protection assay. At least 3 animals were used for each group in all experiments. Values were corrected for 18S RNA; P < 0.05 for all TNF-alpha treatments. TNF-alpha decreased endogenous collagen-alpha 1(I) mRNA in the liver; P < 0.05. C: representative examples of RNase protection assay showing expression of hGH transgenes in control (lanes 1, 2, 5, and 6) and TNF-alpha (lanes 3, 4, 7, and 8)-treated animals bearing -2300 COL-hGH (lanes 1-4) or -440 COL-hGH minigenes (lanes 5-8). Protected hGH mRNA segment is 168 nucleotides.

Because TNF-alpha could affect liver collagen-alpha 1(I) gene expression directly and/or indirectly through induction of cytokine cascades in other cells, we analyzed the effects of TNF-alpha on cis-regulatory regions of the collagen-alpha 1(I) gene in cultured cells. Stellate cells were freshly isolated from -440 COL-hGH transgenic mice, and to prevent their activation and proliferation (27, 32) after enzymatic isolation, cells were cultured with or without treatment for 6 days on an EHS matrix. Under these conditions, cell quiescence was documented by the number of cells in S phase (32) and by [3H]thymidine incorporation (27). In addition, stellate cells cultured on an EHS matrix did not express alpha -SMA whether or not they were treated with TNF-alpha (Fig. 3A, left and middle). In contrast, stellate cells were activated when treated with FeSO4 (50 µM)/ascorbate (200 µM) for 6 days (Fig. 3A, right), which induces oxidative stress as described previously (32). Because primary quiescent stellate cells have a low collagen gene expression (26), we treated these cells with TNF-alpha for 6 days to facilitate an accurate determination of the cytokine effects. Treatment of stellate cells for 6 days with TNF-alpha (10 ng/ml) inhibited expression of the -440 COL-hGH transgene to an extent comparable to that induced by TNF-alpha in animals bearing this collagen-hGH transgene (Fig. 3, B and C). Values for hGH mRNA were normalized by the internal standard 18S RNA as described previously (26).


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Fig. 3.   TNF-alpha inhibits expression of human collagen-alpha 1(I)-hGH minigenes in hepatic stellate cells. A: alpha -smooth muscle actin (alpha -SMA) expression in hepatic stellate cells. Hepatic stellate cells were freshly isolated from transgenic animals and cultured on EHS matrix. Representative examples of untreated control (left) or stellate cells treated with either TNF-alpha (10 ng/ml; middle) or FeSO4 (50 µM)/ascorbate (20 mM) (right) are shown. Day 6 cells were fixed with acetone and methanol (60:40) and immunofluorescence for alpha -SMA was performed using specific antibodies as described previously (32). Hoechst-33342 was used as nuclear counterstain. alpha -SMA expression was negative in control or TNF-alpha -treated cells cultured on EHS matrix, whereas Fe2+/ascorbate stimulated alpha -SMA expression. B: collagen-hGH transgene expression in primary cell cultures. Hepatic stellate cells were freshly isolated from transgenic animals and cultured on EHS matrix as described in METHODS. Treatment with TNF-alpha (10 ng/ml) decreased hGH expression in stellate cells as measured by RNase protection assay from triplicate samples; P < 0.05 for TNF-alpha treatment. Values of hGH mRNA were corrected by internal standard 18S RNA. C: representative examples of RNase protection assay for hepatic stellate cells isolated from -440 COL-hGH transgenic animals showing protected 168 nucleotide hGH band in control (lanes 1 and 2) and TNF-alpha -treated (lanes 3 and 4) cells.

The promoter and first intron of the collagen-alpha 1(I) gene are highly conserved among different species, including humans, mice, and rats (15, 35, 43). Therefore, additional characterization of the cis-regulatory region within the collagen 5' flanking sequences was obtained by transfecting LUC chimeric reporter genes driven by segments of the mouse collagen-alpha 1(I) gene into day 10 stellate cells growing on a collagen type I matrix. Using a transfection-enhancing reagent and lipofectamine, we achieved a high-efficiency transfection for activated stellate cells growing on a collagen type I matrix as described previously (27). To obtain optimal reporter expression, cells were harvested 48 h after transfection. Expression of the LUC reporter containing 2,300 bp or 220 bp of the 5' flanking region of the mouse alpha 1(I) collagen gene (without the first intron) was inhibited by TNF-alpha (Fig. 4). The transfection efficiency, as determined with a pLUC vector, was essentially identical in control and TNF-alpha -treated cells (13.6 ± 1.5 vs. 14.1 ± 2.5 U/mg DNA; not significant), indicating that TNF-alpha did not decrease collagen-chimeric reporter gene expression spuriously by inhibiting the transfection of these genes.


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Fig. 4.   TNF-alpha inhibits transcription from promoter of collagen-alpha 1(I) gene. Cells were transfected for 48 h with chimeric reporter genes as described in METHODS. pCol2LUC and pCol3LUC contain a fragment of mouse collagen type I promoter (-2300/+110 bp and -220/+110 bp, respectively), whereas pLUC does not. Luciferase assay was performed as described by the manufacturer, corrected for cellular DNA content. Forty-eight-hour treatment with TNF-alpha (10 ng/ml) decreased pCol2LUC (n:6) and pCol3LUC (n:8) expression compared with control; P < 0.05.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

We (50) and others (2) have suggested that TNF-alpha could play an important role in tissue fibrogenesis by inhibiting collagen-alpha 1(I) gene transcription. This study supports the hypothesis that TNF-alpha inhibits liver collagen-alpha 1(I) gene expression independent of the confounding variables of tissue necrosis and inflammation. In addition, we demonstrated that TNF-alpha inhibits collagen-alpha 1(I) gene expression in cultured stellate cells, independent of the confounding variables of stellate cell activation or proliferation (16, 20, 21). Although an increase in NF1 (26) and Sp1 (44) binding activities has been reported in stellate cells after their activation, these changes appear to be predominantly quantitative (26, 44).

In this report we show that chronically elevated serum TNF-alpha inhibits liver collagen-alpha 1(I) gene expression, before the onset of weight loss in these animals (5, 7, 8). To eliminate the confounding variable of weight loss, which is known to downregulate collagen-alpha 1(I) gene expression in scorbutic and fasted animals (12, 13, 40, 51), we studied TNF-alpha -secreting precachectic animals (5, 7, 8). The body weight of precachectic TNF-alpha animals was essentially equal to that of CHO mice. Here we show that TNF-alpha decreases the steady-state level of collagen-alpha 1(I) mRNA and collagen synthesis in the liver of precachectic mice, in agreement with the transcriptional inhibition induced by TNF-alpha in cultured human and murine fibroblasts (50). Similar effects on liver collagen-alpha 1(I) gene expression were observed after only an 8-h treatment with TNF-alpha , in agreement with the rapid effects of this cytokine on cultured cells (2, 50).

To characterize the regulatory region of the collagen-alpha 1(I) gene responsive to the inhibitory effects of TNF-alpha , we analyzed the expression of reporter chimeric genes in transgenic mice. Although during CCl4-induced fibrogenesis in the liver expression of the collagen-alpha 1(I) gene requires only the presence of an upstream -440 bp region (26), inhibitory cis elements could be localized in any region of the gene, including 5', intronic, and 3' segments (11, 19). We found that TNF-alpha inhibits expression of the -440 COL-hGH as well as of the -2300 COL-hGH transgene in the liver, indicating that the inhibitory effect of this cytokine is exerted on the -440 bp of the 5' flanking region and/or the first intron of the collagen-alpha 1(I) gene. TNF-alpha also inhibited collagen synthesis and gene expression in the skin of precachectic animals; however, this effect required a region of the collagen-alpha 1(I) gene beyond -440 bp (8), suggesting a different inhibition of collagen-alpha 1(I) gene expression by TNF-alpha in various target tissues. Interestingly, the stimulation of collagen-alpha 1(I) transcription is also regulated in a tissue-specific manner. The -440 bp region is sufficient in the liver, but the region between -440 bp and -2,300 bp is required for the efficient transcription of this gene in skin and tendon (26). To our knowledge, this is the first evidence of a tissue-specific inhibitory mechanism in gene transcription.

Inagaki et al. (29) have identified a TNF-alpha -responsive element within the -378 to -235 sequence (TbRE) of the collagen alpha 2(I) gene in dermal fibroblasts. The suppressive effect of TNF-alpha on collagen-alpha 2(I) expression was not mediated by either AP-1 or nuclear factor-kappa beta . Interestingly, transforming growth factor-beta (TGF-beta ) stimulates collagen-alpha 2(I) gene transcription through increasing the binding of a Sp1-containing complex to the same cis-acting TbRE region (28). It has been suggested that an NF1 binding site mediates the transcriptional activation of collagen-alpha 2(I) gene by TGF-beta (47). It remains to be determined whether similar convergency of the TGF-beta and TNF-alpha pathways occurs in the collagen-alpha 1(I) gene in hepatic stellate cells. However, in fibroblasts the TGF-beta -responsive cis element of the collagen-alpha 1(I) gene (alpha 1-TAE) is present within the -1627 to -1643 bp region of the gene (45) that is required for responsiveness to TNF-alpha in the skin (8). The activation of collagen-alpha 1(I) gene transcription of TGF-beta through the alpha 1-TAE site is independent of NF1 or AP-2 proteins (46).

Although TNF-alpha inhibits collagen gene expression (2, 50) and stimulates collagenase activity (17) and collagenase gene expression (6) in cultured cells, TNF-alpha plays a key role in the development of lung fibrosis induced by silica or bleomycin (41, 42). Moreover, expression of a TNF-alpha transgene in murine lung causes progressive pulmonary fibrosis (38). The apparent discrepancy of these results could be explained in different ways. First, the models of lung fibrosis are characterized by the development of necrosis and inflammation (38, 41, 42). In our study, the effects of TNF-alpha on stellate cell collagen gene expression were analyzed independent of necroinflammatory reactions. In support of our results, neither the proliferation nor the activation of primary rat stellate cells, features associated with their collagen production (26, 36, 38, 41), were affected by TNF-alpha (24). The number of cells remained essentially unchanged after 48 h in control and TNF-alpha -treated cultures (data not shown).

Another mechanism by which TNF-alpha could contribute to tissue fibrosis is the inhibition of collagen phagocytosis mediated by decreased collagen binding to the alpha 2beta 1-integrin receptor (14, 37). Perhaps the nature of a given pathological condition may determine the overall effect of TNF-alpha on tissue fibrosis. In the presence of intense necroinflammatory reactions, TNF-alpha could stimulate, directly or indirectly, tissue fibrosis. In this context, induction of an acute phase response stimulates collagen gene expression and this effect was blocked by interleukin-6 (IL-6) antibodies (23). Although TNF-alpha could stimulate IL-6 synthesis, TNF-alpha has many other independent effects (4). Furthermore, we have shown that the inhibition of collagen gene expression in cultured human fibroblasts by TNF-alpha is not mediated by IL-6 (50). From our study, it can also be concluded that p55 TNF-alpha 1 receptor (TNFR1) activation is sufficient to mediate the effects of TNF-alpha on collagen-alpha 1(I) gene expression, because human TNF-alpha , used in our experiments, interacts with the murine TNFR1 but not, apparently, with the murine TNFR2 receptor (33).

Because in the intact animal TNF-alpha could modulate liver collagen-alpha 1(I) gene expression indirectly through cytokine cascades (4, 53), we tested whether the same cis-regulatory region of the collagen-alpha 1(I) gene contains the TNF-alpha -responsive element(s) in primary stellate cell cultures. In primary cultures of quiescent stellate cells (to avoid the confounding variables of cell activation and cell proliferation) bearing the -440 COL-hGH transgene, TNF-alpha inhibited the expression of the hGH reporter to a degree similar to that observed in the -440 COL-hGH transgenic mice. These studies indicate that the -440 bp segment of the 5' flanking region and/or the first intron contains the TNF-alpha -responsive element(s). Further characterization of the sequences responsive to TNF-alpha was obtained by transfection into stellate cells of a chimeric LUC reporter gene. A regulatory region of the collagen-alpha 1(I) gene containing the -220/+110 bp segment in the absence of the first intron was sufficient for the inhibition of LUC reporter expression by TNF-alpha in transfected primary stellate cells. Whether TNF-alpha inhibits hepatic collagen gene expression and interferes with liver repair (30) in patients with chronic diseases of the liver and other tissues associated with higher levels of serum TNF-alpha (4, 22, 36, 48, 52, 54) cannot be inferred from our study.

Taken together, these studies suggest that TNF-alpha inhibits collagen-alpha 1(I) gene expression by affecting directly or indirectly the interaction of transcriptional factors within the -440 bp of the collagen-alpha 1(I) promoter and/or first intron in hepatic stellate cells in vivo and in culture. In conclusion, our studies suggest that TNF-alpha affects liver collagen-alpha 1(I) gene expression through a tissue-specific region of this gene different from that required to inhibit collagen gene expression in the skin (8).

    ACKNOWLEDGEMENTS

We thank K. Pak for technical assistance and L. Masse for the preparation of this manuscript. We are indebted to Dr. P. Bornstein (University of Washington, Seattle) for providing the founder transgenic mice for lines -2300 COL-hGH and -440 COL-hGH; to Dr. A. Oliff (Merck Research Laboratories, West Point, PA) for the CHO and TNF-alpha cells; and to Dr. K. Roebuck for assistance in the RNase protection assay.

    FOOTNOTES

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38652 and DK-46971 and National Institute of General Medical Sciences Grant GM-47165 and by grants from the Department of Veterans Affairs, the American Liver Foundation, and the Ford Foundation. D. J. Kim was supported by a grant from the IL-SONG Education Foundation, South Korea.

K. Houglum and M. Buck contributed equally to this study.

Address for reprint requests: M. Chojkier, Dept. of Medicine and Center for Molecular Genetics (9-111D), Univ. of California, San Diego, CA 92161.

Received 4 August 1997; accepted in final form 15 January 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 274(5):G840-G847