Department of Medicine, Veterans Affairs Medical Center, and Center for Molecular Genetics, University of California, San Diego, California 92161
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although tumor
necrosis factor- (TNF-
) inhibits
collagen-
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-
affects constitutive hepatic
collagen metabolism in vivo by inoculating nude mice with Chinese
hamster ovary (CHO) cells secreting TNF-
(TNF-
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-
mice. In transgenic mice, after 8 h, TNF-
(500 ng
at 0 and 5 h) inhibited the liver expression of the
collagen-
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-
1(I)-hGH transgene was
responsive to TNF-
treatment independent of the activation of these
cells. Transfection studies in stellate cells allowed further
characterization of this TNF-
-responsive segment to
220 bp of
the 5' region. Because in the skin the inhibitory effect of
TNF-
involves a regulatory region of the
collagen-
1(I) gene beyond
440 bp, we herein identify a novel tissue-specific regulation of
collagen-
1(I) gene by TNF-
.
liver fibrosis; cytokines; cachexia; transcription; stellate cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TUMOR NECROSIS FACTOR- (TNF-
) is secreted by
macrophages in response to inflammation, infection, and cancer (4).
TNF-
plays a major part in tissue inflammation (53) and remodeling by stimulating production of collagenase gene expression (6). Also,
TNF-
inhibits collagen-
1(I)
gene expression in cultured fibroblasts (50) and in activated hepatic
stellate cells (lipocytes or Ito cells) (2). In contrast,
TNF-
has been implicated as a profibrotic agent in lung inflammation
and fibrosis induced by silica or bleomycin (41, 42), and expression of
a TNF-
transgene in murine lung causes fibrosing alveolitis (38).
These data suggest that the effects of TNF-
on fibrogenesis may be different in various pathological conditions, such as those associated with lung inflammation (41, 42).
Although elevated serum TNF- 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-
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-
inhibit constitutive liver
collagen-
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-
1(I) gene were
sufficient for responsiveness to TNF-
treatment. Furthermore,
transfection of chimeric reporter genes to hepatic stellate cells
allowed us to circumscribe the TNF-
responsiveness to the
220
bp 5' region. Because in the skin the inhibitory effect of
TNF-
involves a regulatory region of the
collagen-
1(I) gene beyond
440 bp (8), we herein identify a novel tissue-specific
regulation of collagen-
1(I) gene by TNF-
.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse model of cachexia.
Chinese hamster ovary (CHO) cells were stably transfected with either
the human TNF- gene cloned into a mammalian expression vector
(TNF-
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-
cells, but not the CHO cells, produced TNF-
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-
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-
mice). Serum TNF-
levels were determined
by an immunoassay using monoclonal antibodies against human TNF-
(Endogen) (5, 7). In other experiments, adult C57BL/6 mice were
injected intraperitoneally with either 500 ng of human recombinant
TNF-
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 atSDS-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-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-
1(I), and
2-microglobulin (26, 39, 49).
Collagen-
1(I) mRNA and hGH mRNA
values were measured simultaneously with the internal standards
2-microglobulin mRNA and 18S
RNA, respectively, as described by us previously (6-8, 18, 26).
Expression of chimeric
collagen-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
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-
1(I) gene responsive to TNF-
was characterized in transgenic mice expressing hGH under the direction of
2,300 bp or
440 bp of the human
collagen-
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 -smooth muscle
actin (
-SMA), judging by the immunofluorescence using specific
antibodies against
-SMA (Sigma) (32).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two weeks after their inoculation with TNF- cells, mice had serum
TNF-
concentrations of 275 ± 75 pg/ml as measured by a specific
immunoassay, comparable to those reported previously (5, 7, 8). These
serum TNF-
concentrations are lower than those utilized in
experiments with cultured fibroblasts and hepatic stellate cells (2,
50). More importantly, similar or higher TNF-
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-
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-
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-
mice compared with CHO mice (Table
1). The specific activity of total
noncollagen proteins in the liver was not affected in the precachectic
TNF-
mice (Table 1), in agreement with studies in cultured
fibroblasts (50). However, TNF-
also inhibits albumin gene
expression in precachectic mice (5). These findings indicate that
TNF-
inhibits collagen type I production independent of factors
related to weight loss (12, 13, 40). The purity of the extracted liver
collagen in TNF-
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-
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).
|
|
To determine whether the decreased collagen synthesis in the liver of
precachectic TNF- mice was the result of an inhibition of
collagen-
1(I) gene expression,
we analyzed collagen mRNA levels. Total RNA was purified from the liver
of TNF-
and CHO mice, and the abundance of
collagen-
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-
1(I) mRNA was
110 bases, confirming the specificity of the assay. The levels of
collagen-
1(I) mRNA (corrected
by the steady-state levels of
2-microglobulin mRNA) were
decreased in the liver of TNF-
mice (Fig. 1,
B and
C), indicating a selective effect of
chronically elevated TNF-
on
collagen-
1(I) gene expression in the liver, similar to that found in cultured fibroblasts and stellate cells (2, 50).
To characterize the TNF--responsive region within the
collagen-
1(I) gene, we
performed experiments with transgenic mice expressing the hGH reporter
under the direction of regulatory regions of the human
collagen-
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-
results in a peak
TNF-
serum level of ~20 ng/ml at 45 min, decreasing to ~4 ng/ml
at 3 h (1). These TNF-
values are comparable to those found in
cachectic TNF-
mice (5, 7, 8) and in cachectic patients (22, 48,
54). Both the hGH transgene mRNA and the endogenous
collagen-
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-
treatment in the
same transgenic line (see Figs. 2 and 3). TNF-
inhibits
collagen-
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-
(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).
|
Because TNF- could affect liver
collagen-
1(I) gene expression
directly and/or indirectly through induction of cytokine
cascades in other cells, we analyzed the effects of TNF-
on
cis-regulatory regions of the
collagen-
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
-SMA whether or not they were treated with
TNF-
(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-
for 6 days to facilitate an accurate determination of the
cytokine effects. Treatment of stellate cells for 6 days with TNF-
(10 ng/ml) inhibited expression of the
440 COL-hGH transgene to
an extent comparable to that induced by TNF-
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).
|
The promoter and first intron of the
collagen-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-
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
1(I) collagen gene
(without the first intron) was inhibited by TNF-
(Fig.
4). The transfection efficiency, as
determined with a pLUC vector, was essentially identical in control and
TNF-
-treated cells (13.6 ± 1.5 vs. 14.1 ± 2.5 U/mg DNA; not
significant), indicating that TNF-
did not decrease collagen-chimeric reporter gene expression spuriously by inhibiting the
transfection of these genes.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We (50) and others (2) have suggested that TNF- could play an
important role in tissue fibrogenesis by inhibiting
collagen-
1(I) gene
transcription. This study supports the hypothesis that TNF-
inhibits
liver collagen-
1(I) gene
expression independent of the confounding variables of tissue necrosis
and inflammation. In addition, we demonstrated that TNF-
inhibits collagen-
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- inhibits
liver collagen-
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-
1(I) gene
expression in scorbutic and fasted animals (12, 13, 40, 51), we studied
TNF-
-secreting precachectic animals (5, 7, 8). The body weight of
precachectic TNF-
animals was essentially equal to that of CHO mice.
Here we show that TNF-
decreases the steady-state level of
collagen-
1(I) mRNA and collagen
synthesis in the liver of precachectic mice, in agreement with the
transcriptional inhibition induced by TNF-
in cultured human and
murine fibroblasts (50). Similar effects on liver
collagen-
1(I) gene expression
were observed after only an 8-h treatment with TNF-
, in agreement
with the rapid effects of this cytokine on cultured cells (2, 50).
To characterize the regulatory region of the
collagen-1(I) gene responsive
to the inhibitory effects of TNF-
, we analyzed the expression of
reporter chimeric genes in transgenic mice. Although during
CCl4-induced fibrogenesis in the
liver expression of the
collagen-
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-
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-
1(I) gene. TNF-
also inhibited collagen synthesis and gene expression in the skin of
precachectic animals; however, this effect required a region of the
collagen-
1(I) gene beyond
440 bp (8), suggesting a different inhibition of
collagen-
1(I) gene expression
by TNF-
in various target tissues. Interestingly, the stimulation of
collagen-
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--responsive element within
the
378 to
235 sequence (TbRE) of the collagen
2(I) gene in dermal
fibroblasts. The suppressive effect of TNF-
on collagen-
2(I) expression was
not mediated by either AP-1 or nuclear factor-
. Interestingly,
transforming growth factor-
(TGF-
) stimulates
collagen-
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-
2(I) gene by TGF-
(47). It remains to be determined whether similar convergency of the
TGF-
and TNF-
pathways occurs in the
collagen-
1(I) gene in hepatic
stellate cells. However, in fibroblasts the TGF-
-responsive cis element of the
collagen-
1(I) gene
(
1-TAE) is present within the
1627 to
1643 bp region of the gene (45) that is required for responsiveness to TNF-
in the skin (8). The activation of
collagen-
1(I) gene
transcription of TGF-
through the
1-TAE site is
independent of NF1 or AP-2 proteins (46).
Although TNF- inhibits collagen gene expression (2, 50) and
stimulates collagenase activity (17) and collagenase gene expression
(6) in cultured cells, TNF-
plays a key role in the development of
lung fibrosis induced by silica or bleomycin (41, 42). Moreover,
expression of a TNF-
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-
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-
(24). The number of cells remained essentially
unchanged after 48 h in control and TNF-
-treated cultures (data not
shown).
Another mechanism by which TNF- could contribute to tissue fibrosis
is the inhibition of collagen phagocytosis mediated by decreased
collagen binding to the
2
1-integrin
receptor (14, 37). Perhaps the nature of a given pathological condition
may determine the overall effect of TNF-
on tissue fibrosis. In the presence of intense necroinflammatory reactions, TNF-
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-
could stimulate IL-6 synthesis,
TNF-
has many other independent effects (4). Furthermore, we have
shown that the inhibition of collagen gene expression in cultured human fibroblasts by TNF-
is not mediated by IL-6 (50). From our study, it
can also be concluded that p55 TNF-
1 receptor (TNFR1) activation is sufficient to mediate the effects of TNF-
on
collagen-
1(I) gene expression,
because human TNF-
, used in our experiments, interacts with the
murine TNFR1 but not, apparently, with the murine TNFR2 receptor (33).
Because in the intact animal TNF- could modulate liver
collagen-
1(I) gene expression
indirectly through cytokine cascades (4, 53), we tested whether the
same cis-regulatory region of the
collagen-
1(I) gene contains the
TNF-
-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-
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-
-responsive element(s). Further characterization of
the sequences responsive to TNF-
was obtained by transfection into
stellate cells of a chimeric LUC reporter gene. A regulatory region of
the collagen-
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-
in transfected primary stellate cells. Whether TNF-
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-
(4, 22, 36, 48, 52, 54)
cannot be inferred from our study.
Taken together, these studies suggest that TNF- inhibits
collagen-
1(I) gene expression
by affecting directly or indirectly the interaction of transcriptional
factors within the
440 bp of the
collagen-
1(I) promoter
and/or first intron in hepatic stellate cells in vivo and in
culture. In conclusion, our studies suggest that TNF-
affects liver
collagen-
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-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alcorn, J. M.,
J. Fierer,
and
M. Chojkier.
The acute-phase response protects mice from D-galactosamine sensitization to endotoxin and tumor necrosis factor-.
Hepatology
15:
122-129,
1992[Medline].
2.
Armendariz-Borunda, J.,
K. Katayama,
and
J. M. Seyer.
Transcriptional mechanisms of type I collagen gene expression are differentially regulated by interleukin-1, tumor necrosis factor-
, and transforming growth factor in Ito cells.
J. Biol. Chem.
267:
14316-14321,
1992
3.
Bedossa, P.,
K. Houglum,
C. Trautwein,
A. Holstege,
and
M. Chojkier.
Stimulation of collagen 1(I) gene expression is associated with lipid peroxidation in hepatocellular injury. A link to tissue fibrosis?
Hepatology
15:
1262-1271,
1994.
4.
Beutler, B.
Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine. New York: Raven, 1992.
5.
Brenner, D. A.,
M. Buck,
S. P. Feitelberg,
and
M. Chojkier.
Tumor necrosis factor- inhibits albumin gene expression in a murine model of cachexia.
J. Clin. Invest.
85:
248-255,
1990[Medline].
6.
Brenner, D. A.,
M. O'Hara,
P. Angel,
M. Chojkier,
and
M. Karin.
Prolonged activation of jun and collagenase genes by tumor necrosis factor-.
Nature
337:
661-663,
1989[Medline].
7.
Buck, M.,
and
M. Chojkier.
Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants.
EMBO J.
15:
1753-1765,
1996[Abstract].
8.
Buck, M.,
K. Houglum,
and
M. Chojkier.
Tumor necrosis factor- inhibits collagen
1(I) gene expression and wound healing in a murine model of cachexia.
Am. J. Pathol.
149:
195-204,
1996[Abstract].
9.
Buck, M.,
H. Turler,
and
M. Chojkier.
LAP (NF-IL6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation.
EMBO J.
13:
851-860,
1994[Abstract].
10.
Chojkier, M.
Hepatocyte collagen production in vivo in normal rats.
J. Clin. Invest.
78:
333-339,
1986[Medline].
11.
Chojkier, M.
Regulation of liver-specific gene expression.
In: Progress in Liver Diseases, edited by J. Boyer,
and R. Ockner. Orlando, FL: Saunders, 1995, p. 37-61.
12.
Chojkier, M.,
M. Flaherty,
B. Peterkofsky,
G. H. Majmudar,
R. G. Spanheimer,
and
D. A. Brenner.
Different mechanisms decrease hepatic collagen and albumin production in fasted rats.
Hepatology
8:
1040-1045,
1988[Medline].
13.
Chojkier, M.,
R. Spanheimer,
and
B. Peterkofsky.
Specifically decreased collagen biosynthesis in scurvy dissociated from an effect on proline hydroxylation and correlated with body weight loss.
J. Clin. Invest.
72:
826-835,
1983[Medline].
14.
Chou, D. H.,
W. Lee,
and
C. A. G. McCulloch.
TNF- inactivation of collagen receptors.
J. Immunol.
156:
4354-4362,
1996[Abstract].
15.
Chu, M.-L.,
W. deWet,
M. Bernard,
and
F. Ramirez.
Fine structural analysis of the human pro-1(I) collagen gene.
J. Biol. Chem.
260:
2315-2320,
1985[Abstract].
16.
Davis, B. H.,
B. M. Pratt,
and
J. A. Madri.
Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels.
J. Biol. Chem.
262:
10280-10286,
1987
17.
Dayer, J. M.,
B. Beutler,
and
A. Cerami.
Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts.
J. Exp. Med.
162:
2163-2168,
1985[Abstract].
18.
Descombes, P.,
M. Chojkier,
S. Lichtsteiner,
E. Falvey,
and
U. Schibler.
LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein.
Genes Dev.
4:
1541-1551,
1990[Abstract].
19.
Falvey, E.,
and
U. Schibler.
How are the regulators regulated?
FASEB J.
5:
309-314,
1991
20.
Friedman, S. L.,
F. J. Roll,
J. Boyles,
D. M. Arenson,
and
D. M. Bissell.
Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix.
J. Biol. Chem.
264:
10756-10762,
1989
21.
Geerts, A.,
P. De Bleser,
M. L. Hautekeete,
T. Niki,
and
E. Wisse.
Fat-storing (Ito) cell biology.
In: The Liver: Biology and Pathobiology, edited by I. M. Arias,
T. Boyer,
N. Fausto,
W. Jakoby,
D. Schachter,
and D. A. Shafritz. New York: Raven, 1994, p. 819-838.
22.
Grau, G. E.,
T. E. Taylor,
M. E. Molyneux,
J. J. Wirima,
P. Vassalli,
M. Hommel,
and
P. Lambert.
Tumor necrosis factor and disease severity in children with Falciparum malaria.
N. Engl. J. Med.
320:
1586-1591,
1989[Abstract].
23.
Greenwel, P.,
M. J. Iraburu,
M. Reyes-Romero,
N. Merza-Cruz,
E. Casado,
J. A. Solis-Herruzo,
and
M. Rojkind.
Induction of an acute phase response in rats stimulates the expression of 1(1) procollagen messenger ribonucleic acid in their livers.
Lab. Invest.
72:
83-91,
1995[Medline].
24.
Gressner, A. M.,
B. Lahme,
and
A. Brenzel.
Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cells.
Hepatology
22:
1507-1518,
1995[Medline].
25.
Houglum, K.,
M. Buck,
V. Adir,
and
M. Chojkier.
LAP (NF-IL6) transactivates the collagen 1(I) gene from a 5' regulatory region.
J. Clin. Invest.
94:
808-814,
1994[Medline].
26.
Houglum, K.,
M. Buck,
J. Alcorn,
S. Contreras,
P. Bornstein,
and
M. Chojkier.
Two different cis-acting regulatory regions direct cell-specific transcription of the collagen 1(I) gene in hepatic stellate cells and in skin and tendon fibroblasts.
J. Clin. Invest.
96:
2269-2276,
1995[Medline].
27.
Houglum, K.,
K. S. Lee,
and
M. Chojkier.
Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on Serine 133.
J. Clin. Invest.
99:
1322-1389,
1997
28.
Inagaki, Y.,
S. Truter,
and
F. Ramirez.
Transforming growth factor- stimulates
2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site.
J. Biol. Chem.
269:
14828-14834,
1994
29.
Inagaki, Y.,
S. Truter,
S. Tanaka,
M. DiLiberto,
and
F. Ramirez.
Overlapping pathways mediate the opposing actions of tumor necrosis factor- and transforming growth factor-
on
2(I) collagen gene transcription.
J. Biol. Chem.
270:
3353-3358,
1995
30.
Ingber, D. E.
Extracellular matrix and the development of tissue architecture: a mechanochemical perspective.
In: Extracellular Matrix, edited by M. Zern,
and L. Reid. New York: Dekker, 1993, p. 403-428.
31.
Lahdevirta, J.,
C. P. Maury,
A. Teppo,
and
H. Repo.
Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome.
Am. J. Med.
85:
289-291,
1988[Medline].
32.
Lee, K. S.,
M. Buck,
K. Houglum,
and
M. Chojkier.
Activation of hepatic stellate cells by TGF- and collagen type I is mediated by oxidative stress through c-myb expression.
J. Clin. Invest.
96:
2461-2468,
1995[Medline].
33.
Lewis, M.,
L. A. Tartaglia,
A. Lee,
G. L. Bennett,
G. C. Rice,
G. H. W. Wong,
E. Y. Chen,
and
D. V. Goeddel.
Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific.
Proc. Natl. Acad. Sci. USA
88:
2830-2834,
1991[Abstract].
34.
Liska, D. J.,
M. J. Reed,
E. H. Sage,
and
P. Bornstein.
Cell-specific expression of 1(I) collagen-hGH minigenes in transgenic mice.
J. Cell Biol.
125:
695-704,
1994[Abstract].
35.
Liska, D. J.,
J. L. Slack,
and
P. Bornstein.
A highly conserved intronic sequence is involved in transcriptional regulation of the 1(I) collagen gene.
Cell Regul.
1:
487-498,
1990[Medline].
36.
McClain, C.,
D. Hill,
J. Schmidt,
and
A. M. Diehl.
Cytokines and alcoholic liver disease.
Semin. Liver Dis.
13:
170-182,
1993[Medline].
37.
McCulloch, C. A. G.,
and
G. C. Knowles.
Deficiencies in collagen phagocytosis by human fibroblasts in vitro: a mechanism for fibrosis?
J. Cell. Physiol.
155:
461-471,
1993[Medline].
38.
Miyazaki, Y.,
K. Araki,
C. Vesin,
I. Garcia,
Y. Kapanci,
J. A. Whitsett,
P. F. Piguet,
and
P. Vassalli.
Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis. A mouse model of progressive pulmonary fibrosis.
J. Clin. Invest.
96:
250-259,
1995[Medline].
39.
Parnes, J. R.,
and
J. G. Seidman.
Structure of wild-type and mutant mouse 2-microglobulin genes.
Cell
29:
661-669,
1982[Medline].
40.
Peterkofsky, B.,
R. G. Spanheimer,
and
T. A. Bird.
Coordinate regulation of cartilage collagen and proteoglycan synthesis in scorbutic guinea pig.
In: Development and Diseases of Cartilage and Bone Matrix, edited by A. Sen,
and T. Phornhill. New York: Liss, 1987, p. 33-43.
41.
Piguet, P. F.,
M. A. Collart,
G. E. Grau,
Y. Kapanci,
and
P. Vassalli.
Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis.
J. Exp. Med.
170:
655-663,
1989[Abstract].
42.
Piguet, P. F.,
M. A. Collart,
G. E. Grau,
A. P. Sappino,
and
P. Vassalli.
Requirement of tumor necrosis factor for development of silica-induced pulmonary fibrosis.
Nature
344:
245-247,
1990[Medline].
43.
Ravazzolo, R.,
G. Karsenty,
and
B. de Crombrugghe.
A fibroblast-specific factor binds to an upstream negative control element in the promoter of the mouse 1(I) collagen gene.
J. Biol. Chem.
266:
7382-7387,
1991
44.
Rippe, R. A.,
G. Almounajed,
and
D. Brenner.
Sp1 binding activity increases in activated Ito cells.
Hepatology
22:
241-251,
1995[Medline].
45.
Ritzenthaler, J. D.,
R. H. Goldstein,
A. Fine,
A. Lichtler,
D. W. Rowe,
and
B. D. Smith.
Transforming-growth-factor- activation elements in the distal promoter regions of the rat
1 type I collagen gene.
Biochem. J.
280:
157-162,
1991[Medline].
46.
Ritzenthaler, J. D.,
R. H. Goldstein,
A. Fine,
and
B. D. Smith.
Regulation of the 1(I) collagen promoter via a transforming growth factor-
activation element.
J. Biol. Chem.
268:
13625-13631,
1993
47.
Rossi, P.,
G. Karsenty,
A. B. Roberts,
N. S. Roche,
M. B. Sporn,
and
B. de Crombrugghe.
A nuclear factor I binding site mediates the transcriptional activation of a type I collagen promotor by transforming growth factor-.
Cell
52:
405-414,
1988[Medline].
48.
Scuderi, P.,
K. S. Lam,
K. J. Ryan,
E. Peterson,
K. E. Sterling,
P. R. Finley,
C. G. Ray,
D. J. Slymen,
and
S. E. Salmon.
Raised serum levels of tumor necrosis factor in parasitic infections.
Lancet
2:
1364-1365,
1986[Medline].
49.
Slack, J. L.,
D. J. Liska,
and
P. Bornstein.
An upstream regulatory region mediates high-level, tissue-specific expression of the human 1(I) collagen gene in transgenic mice.
Mol. Cell. Biol.
11:
2066-2074,
1991[Medline].
50.
Solis-Herruzo, J. A.,
D. A. Brenner,
and
M. Chojkier.
Tumor necrosis factor- inhibits collagen gene transcription and collagen synthesis in cultured human fibroblasts.
J. Biol. Chem.
263:
5841-5845,
1988
51.
Spanheimer, R. G.,
and
B. Peterkofsky.
A specific decrease in collagen synthesis in acutely fasted, vitamin C-supplemented, guinea pigs.
J. Biol. Chem.
260:
3955-3962,
1985[Abstract].
52.
Tilg, H.,
A. Wilmer,
W. Vogel,
M. Herold,
B. Nölchen,
G. Judmaier,
and
C. Huber.
Serum levels of cytokines in chronic liver diseases.
Gastroenterology
103:
264-274,
1992[Medline].
53.
Tracey, K. J.,
and
A. Cerami.
Tumor necrosis factor, other cytokines and disease.
Annu. Rev. Cell Biol.
9:
317-343,
1993.
54.
Waage, A.,
A. Halstensen,
and
T. Espevik.
Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease.
Lancet
1:
355-357,
1987[Medline].