Regulation of IGF binding protein-1 in Hep G2 cells by
cytokines and reactive oxygen species
Charles H.
Lang,
Gerald J.
Nystrom, and
Robert A.
Frost
Departments of Cellular and Molecular Physiology and Surgery,
Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033
 |
ABSTRACT |
The liver is a
major site of synthesis for insulin-like growth factor binding protein
(IGFBP)-1. Because IGFBP-1 inhibits many anabolic actions of IGF-I,
increases in IGFBP-1 may be partly responsible for the decrease in lean
body mass observed in catabolic/inflammatory conditions. This study
aimed to determine in Hep G2 cells
1) the sensitivity of IGFBP-1
synthesis to treatment with interleukin (IL)-1, tumor necrosis
factor-
(TNF-
), and IL-6, 2)
the ability of reactive oxygen species (ROS) to enhance IGFBP-1
production, and 3) the role of ROS
in mediating cytokine-induced increases in IGFBP-1. Hep G2 cells
responded to IL-1
, TNF-
, and IL-6 with maximal 8- to 10-fold
increases in IGFBP-1 production. Although the maximal responsiveness of
cells treated with TNF-
and IL-6 was 20-30% less than that
with IL-1
, cells demonstrated a similar sensitivity to all cytokines
(half-maximal responsive dose of ~10 ng/ml). A low concentration (3 ng/ml) of all three cytokines had an additive effect on IGFBP-1
production. Cytokines also increased IGFBP-1 mRNA. The half-life of
IGFBP-1 mRNA was ~4 h and not altered by IL-1
. Incubation with
ROS, including
H2O2
and nitric oxide (NO) donors, resulted in a relatively smaller increase
in IGFBP-1. However, preincubating Hep G2 cells with various free
radical scavengers and NO synthase and eicosanoid inhibitors failed to prevent or attenuate cytokine-induced increases in IGFBP-1.
Finally, preincubating cells with pyrrolidinedithiocarbamate (PDTC)
but not SN50 (inhibitors of nuclear factor-
B activation and nuclear translocation, respectively) attenuated increases in IGFBP-1 induced by
IL-1. These results indicate that 1)
proinflammatory cytokines directly enhance IGFBP-1 synthesis by
stimulating transcription without altering mRNA stability,
2) addition of exogenous ROS also
stimulates IGFBP-1 production but to a smaller extent than cytokines,
and 3) the cytokine-induced increase
in IGFBP-1 production is not mediated by endogenous production of ROS
or eicosanoids but appears to at least partially involve a
PDTC-sensitive pathway.
tumor necrosis factor-
; interleukins-1 and -6; free radicals; nitric oxide; nuclear factor-
B; liver; insulin-like growth factor
 |
INTRODUCTION |
THE MAJORITY OF the insulin-like growth factor (IGF)-I
in the blood is carried by high-affinity IGF binding proteins (IGFBPs). Of these binding proteins, only the synthesis and secretion of IGFBP-1
appear to exhibit rapid and dynamic regulation (24). A number of in
vivo and in vitro studies suggest that the liver is the principal site
of synthesis for blood-borne IGFBP-1 (10, 31). Under basal nonstressed
conditions, fluctuation in circulating insulin is believed to be the
primary mechanism by which hepatic IGFBP-1 transcription is regulated
(4, 24, 25, 37). However, during different types of infectious and
inflammatory conditions, changes in the plasma insulin concentration
correlated poorly with the marked elevation of IGFBP-1 observed in the
circulation and in specific tissues (9, 12, 13, 22). Although the physiological importance of this increase is unclear, the majority of
data indicate that elevations in IGFBP-1 impair the anabolic actions of
IGF-I (24). Hence, elevations in IGFBP-1 may be at least partially
responsible for the loss of lean body mass observed in various
inflammatory and catabolic conditions.
Infection and inflammation are known to induce a rapid upregulation of
the synthesis of proinflammatory cytokines by the liver as well as to
increase the concentration of these diverse modulators in the
circulation (8, 15, 17, 47). We have previously demonstrated that
during inflammatory conditions elevations in various cytokines assume a
central role in regulating plasma levels of IGFBP-1. In vivo
administration of tumor necrosis factor-
(TNF-
) or interleukin
(IL)-1
to naive rats produces a rapid and sustained elevation of
IGFBP-1 concentration in blood and liver (9, 13). Furthermore,
pretreatment of rats with neutralizing agents to these cytokines also
partially attenuates the increase in IGFBP-1 produced by gram-negative
infection and endotoxin (11, 22). Finally, preliminary data have shown
that TNF-
, IL-1
, or IL-6 can directly stimulate IGFBP-1 secretion
by the Hep G2 human hepatoma cell line (41), suggesting that at least
part of the in vivo effect of these cytokines is mediated directly at
the level of the hepatocyte.
The whole liver responds to different types of inflammatory stimuli
with a robust generation of reactive oxygen species (ROS), including
hydrogen peroxide
(H2O2)
and nitric oxide (NO) (3, 49). These ROS play an important role in
intracellular killing of bacterial pathogens but when overproduced may
be a contributing factor to hepatic injury and organ dysfunction (18).
The majority of ROS appear to originate from nonparenchymal cells in
the liver (2); however, appropriately stimulated hepatocytes are also capable of producing relatively large amounts of ROS (14, 32). Regardless of the site of synthesis, ROS are now recognized as important intermediates in the intracellular signaling pathways for
various cytokines. However, the role of oxidative stress in mediating
inflammation-induced increases in hepatic IGFBP-1 production has not
been investigated.
The purpose of the present investigation was to determine
1) the sensitivity of IGFBP-1
synthesis in Hep G2 cells treated with TNF-
, IL-1, or IL-6,
2) the ability of ROS to enhance
IGFBP-1 production, and 3) the role
of endogenous ROS in mediating cytokine-induced increases in IGFBP-1.
 |
MATERIALS AND METHODS |
Cell culture.
In general, Hep G2 cells have traditionally been used to elucidate
mechanisms for hormone- and stress-induced changes in IGFBP-1 mRNA and
protein secretion (23, 38) and continue to be used in this regard (6,
45). Specifically, Hep G2 cells were used in the present study because
they are cytokine responsive (30), similar to that observed in isolated
hepatocytes (14). Moreover, these cells and rat H4-II-E hepatoma cells
also have a constitutive secretion of IGFBP-1, and the hormonal
regulation of gene expression is essentially identical to that reported
in hepatocytes (26, 48). Finally, because this cell line is derived
from human cells, the IGFBP-1 secreted into the media can be easily
quantitated by a commercially available immunoradiometric assay (IRMA).
In studies in which the IGFBP-1 concentration was quantitated in the
medium, Hep G2 cells were grown in 24-well plates (Falcon, B-D, Lincoln
Park, NJ). Each well contained 500 µl of MEM (Sigma, St. Louis, MO)
supplemented with 5% calf serum, penicillin (100 U/ml), streptomycin
(100 µg/ml), and amphotericin B (25 µg/ml). Cells were used
5-7 days after subculture, at which time they were near
confluence. On the day of the experiment, the medium was replaced with
serum-free MEM containing 0-300 ng/ml of either recombinant human
(rh) IL-1
(rhIL-1
; gift of Biological Response Modifiers Program, Division of Cancer Treatment/NCI), rhTNF-
(gift
of Amgen, Thousand Oaks, CA), or rhIL-6 (Calbiochem, La Jolla, CA).
After an ~18-h incubation period, culture medium was collected to
determine IGFBP-1 protein concentration. In studies in which IGFBP-1
mRNA abundance was assessed, Hep G2 cells were cultured in 100-mm
plates containing 8 ml of MEM. IGFBP-1 mRNA stability was examined by
incubating control and cytokine-treated cells with the inhibitor of
transcription
5,6-dichloro-
-D-ribofuranosyl-benzimidazole (DRB, 75 µM; Calbiochem) and then quantitating mRNA at various times
thereafter (7). The IGFBP-1 concentration in the conditioned medium
from these cells was also quantitated.
In the second series of studies, the ability of ROS to stimulate
IGFBP-1 secretion was investigated. Cells were incubated for ~18 h
with either
H2O2
or
tert-butyl-H2O2
(100 and 300 µM) or a combination of xanthine (500 µM) and xanthine
oxidase (10 mU/ml). Other cells were incubated with the NO donor sodium
nitroprusside (SNP; 0.1, 0.5, and 1 mM) or
S-nitroso-N-acetylpenicillamine
(SNAP; 0.1, 0.5 and 1 mM). Comparable doses of these ROS donors have been previously demonstrated to stimulate acute phase protein synthesis
and other processes in hepatocytes (20, 40). NO and superoxide anion
can combine to produce peroxynitrite. However, because peroxynitrite
rapidly decomposes, cells were incubated with 3-morpholinosydnonimine
(SIN-1; 500 µM).
In the third series of studies, Hep G2 cells were preincubated for 30 min with one of several oxygen free radical scavengers or inhibitors of
NO or eicosanoid synthesis before addition of cytokine (50 ng/ml). The
oxygen free radical scavengers used included ascorbic acid (1 mM),
-tocopherol (500 µM),
N-acetyl-L-cysteine (NAC; 1 mM), and DMSO (1%). The doses of these scavengers used have
been previously demonstrated to inhibit various cytokine- or
ROS-induced changes in hepatic cells (42, 43). The NO synthesis inhibitors used in this study included
NG-monomethyl-L-arginine
(L-NMMA; 5 mM),
NG-nitro-L-arginine methyl ester
(L-NAME; 5 mM), and
aminoguanidine (0.5 mM). The doses of these inhibitors used have been
previously demonstrated to inhibit cytokine-induced changes in hepatic
cells (16, 21). Inhibitors of eicosanoid biosynthesis were also used
and included the cyclooxygenase inhibitor indomethacin (50 µM) and
the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 µM)
(29). On the basis of measurements of lactate dehydrogenase (LDH) released into the medium, none of the above
treatments at the doses employed resulted in significant cytotoxicity
or overt morphological alterations during the 18-h incubation period
(data not shown).
Both IL-1 and ROS have been demonstrated to induce nuclear factor-
B
(NF-
B) in Hep G2 cells (33, 39), and activation of this DNA-binding
protein has been implicated in mediating a diverse array of biological
responses (1). To determine whether the IL-induced increase in IGFBP-1
was regulated in part by NF-
B activation, cells were pretreated with
pyrrolidinedithiocarbamate (PDTC; 100 µM) 30 min before addition of
IL-1
. In addition, because PDTC does not inhibit NF-
B directly,
Hep G2 cells were incubated with SN50 (18 µM), which is a more
specific cell-permeable peptide inhibitor of NF-
B nuclear
translocation, for 1 h before addition of IL-1
. This peptide
contains the nuclear localization sequence (NLS) for the p50 NF-
B
subunit and the amino-terminal sequence of Kaposi fibroblast growth
factor to promote cell permeability (28).
All reagents were purchased from Sigma Chemical, except the following:
aminoguanidine hemisulfate, SNP, SNAP, and SIN-1 (Research Biochemicals
International, Natick, MA);
L-NMMA and SN50 (Calbiochem); L-NAME (Bachem, Torrance, CA);
and sodium meta-arsenite (Aldrich, St. Louis, MO).
Analytical methods.
Conditioned medium was stored at
20°C until IGFBP-1 could be
quantitated using a two-site IRMA obtained from Diagnostic System Laboratories (Webster, TX).
Briefly, IGFBP-1 mRNA expression was determined as follows. Total RNA
was isolated (TRI Reagent LS, Molecular Research Center, Cincinnati,
OH) from Hep G2 cells, following the manufacturer's protocol. Samples
(10 µg) of total RNA were processed under denaturing conditions on
1% agarose gels containing 6% formaldehyde. Northern blotting was
performed by capillary transfer to Zeta-Probe GT blotting membranes
(Bio-Rad Laboratories, Hercules, CA). A human IGFBP-1 oligonucleotide
probe (Oncogene Research Products, Cambridge, MA) was end-labeled with
[
-32P]ATP
(Amersham, Arlington Heights, IL) using T4 polynucleotide kinase
(Pharmacia Biotech, Piscataway, NJ). For normalization of RNA loading,
a human
-actin oligonucleotide (gift of J. Floros, Pennsylvania
State College of Medicine) was radioactively labeled using
terminal deoxynucleotidyl transferase. Membranes were baked, hybridized, and washed according to the formamide protocol provided with the Zeta-Probe GT. Finally, membranes were exposed to a
PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA), and the
resultant data were quantitated using the accompanying ImageQuant software.
Statistics.
Values are means ± SE (n
5 sets
of duplicate wells for each dose or treatment) or SD
(n = 3 sets of duplicate plates). Data were analyzed by ANOVA, followed by Student-Newman-Keuls test. Statistical significance was set at P < 0.05. The half-maximal responsive dose
(ED50) for IGFBP-1 stimulation
by cytokines was calculated using SigmaPlot (Jandel Scientific
Software, San Rafael, CA).
 |
RESULTS |
Cytokine-induced changes in IGFBP-1.
Figure 1 illustrates that IL-1
produced
a dose-dependent increase in the IGFBP-1 concentration in the medium of
Hep G2 cells incubated for ~18 h. A small (12%), but statistically
significant, increase in IGFBP-1 was detected at a concentration of 1 ng/ml, and a maximal response was observed between 50 and 300 ng/ml. Incubation of cells with IL-1
yielded quantitatively comparable results (data not shown). Stimulation of Hep G2 cells with either TNF-
or IL-6 also produced a dose-dependent increase in IGFBP-1 (Fig. 1). The minimum dose of these cytokines necessary to detect an
increase in IGFBP-1 was 3 ng/ml (P < 0.05), whereas their ability to maximally increase IGFBP-1
concentration was 70-80% of that seen in cells treated with
IL-1
(P < 0.05). However, despite the difference in maximal responsiveness of Hep G2 cells to these cytokines, there was no significant difference in the calculated ED50 for IL-1
(10 ± 3 ng/ml), TNF-
(12 ± 2 ng/ml), and IL-6 (13 ± 3 ng/ml).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Dose-dependent increases in insulin-like growth factor binding protein
(IGFBP)-1 in the culture medium from Hep G2 cells incubated for ~18 h
with either interleukin (IL)-1 , tumor necrosis factor- (TNF- ),
or IL-6. Values are means ± SE of 5-6 sets of duplicate wells
for each dose. Basal (no additions) levels of IGFBP-1 were 24 ± 2, 32 ± 3, and 34 ± 2 ng/ml for plates of cells incubated with
IL-1 , TNF- , or IL-6, respectively. These values were subtracted
from values obtained from cytokine-stimulated cells. There was no
significant difference in the protein concentration per well of cells
during the various experimental conditions (data not shown). Therefore,
similar conclusions are reached regardless of whether data are
expressed as ng/ml or ng/µg cell protein.
|
|
Northern blot analysis revealed that IL-1
increased IGFBP-1 mRNA
expression in a dose-dependent manner in Hep G2 cells incubated for 18 h (Fig. 2,
A and
B). IGFBP-1 mRNA abundance was also
increased in cells incubated with TNF-
and IL-6, but the increment
was smaller than that observed with IL-1
(data not shown). Data from the time course study indicated that IL-1
significantly increased IGFBP-1 mRNA expression by 5 h and that the incremental response was
maximal and stable between 10 and 25 h (Fig.
2C). IL-1
induced comparable
temporal changes in the IGFBP-1 protein concentration in the medium
(data not shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of IL-1 on IGFBP-1 mRNA expression in Hep G2 cells.
A: representative autoradiograph of a
Northern blot, with each lane representing mRNA from a different plate
of cells incubated with no additions (control) or with different doses
of IL-1 (5, 20, or 100 ng/ml). On the basis of densitometric
analysis of human -actin, there was no difference in the amount of
mRNA loaded per lane (data not shown).
B: IGFBP-1 mRNA abundance was
quantitated using a PhosphorImager; data are expressed in arbitrary
volume units (AU). Data were then normalized so that the control value
(0 ng/ml) equaled 1. Values are means ± SD;
n = 3 plates/group.
* P < 0.05 compared with
control value (C). C: temporal changes
in IGFBP-1 mRNA expression in Hep G2 cells in response to IL-1
stimulation. Data are expressed as the ratio of the mRNA abundance for
cells treated with IL-1 (100 ng/ml) divided by the mRNA abundance in
cells incubated in the absence of cytokine for the same length of time.
All values were normalized relative to the amount of -actin (data
not shown). Values are means ± SD;
n = 3 plates/group. All values between
5 and 25 h were significantly higher than values at
time 0 (P < 0.05);
statistics were calculated based on original data.
|
|
To examine IGFBP-1 mRNA stability, cells were incubated overnight with
or without IL-1
. The next morning, all cells received fresh medium
(without IL-1) containing the transcription inhibitor DRB.
RNA was extracted, and then mRNA was quantitated at various time points
thereafter (Fig. 3,
A and
B). DRB effectively reduced IGFBP-1
mRNA in both control and IL-1
-treated cells at each time point
examined, compared with time 0 values.
The calculated apparent half-life for IGFBP-1 mRNA was essentially
identical for control cells and those incubated with IL-1
(3.8 or
4.1 h, respectively). Cells pretreated with IL-1
continued to
synthesize and secrete measurable amounts of IGFBP-1 protein
for the first 10 h after treatment with DRB (Fig.
3C). A negligible amount of IGFBP-1
protein accumulated in the medium between 10 and 15 h, and this was
consistent with the >90% reduction in IGFBP-1 mRNA at the
15-h time point (Fig. 3A). In the
absence of DRB, cells that had been pretreated with IL-1
continued
to secrete IGFBP-1 even though the cytokine had been removed from the
cultures (Fig. 3D). Consistent with its effect on IGFBP-1 mRNA, DRB inhibited IGFBP-1 protein synthesis and
secretion by 85% over a 24-h time period (Fig.
3D).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
A: representative Northern blot of
IGFBP-1 mRNA in Hep G2 cells treated with
5,6-dichloro- -D-ribofuranosyl-benzimidazole
(DRB) without (C) or with IL-1 . Each lane represents mRNA from a
different plate of cells determined at selected times after addition of
DRB (0, 5, 10, 15 h). B: half-life of
IGFBP-1 mRNA in IL-1-treated and untreated (control) cells. Values are
means ± SD; n = 3 plates for each
time point. All data were normalized to the amount of -actin and the
control value at time 0. Where absent,
SD bars are within symbol. C: temporal
changes in IGFBP-1 protein in the medium of Hep G2 cells treated
without (control) or with IL-1 for ~18 h. After this overnight
incubation, fresh medium containing the transcription inhibitor DRB
(but no cytokine) was added to all cells. IGFBP-1 concentration in the
medium was then determined at various times after addition of DRB.
Values are means ± SD of 3 sets of plates. Values with different
letters (a, b, c) are significantly different from each other
(P < 0.05). Where absent, SD bars
are within symbol. D: effect of DRB on
IGFBP-1 protein concentration in conditioned medium of Hep G2 cells
treated with IL-1. Cells were first treated for ~18 h in the presence
or absence of IL-1 . Thereafter, medium was changed and cells were
cultured for 24 h in serum-free medium (containing no IL-1 ) in the
presence or absence of DRB. Cells pretreated with IL-1 (and no DRB)
showed a continued ability to synthesize and secrete IGFBP-1 after
removal of the cytokine, and this response was markedly inhibited by
DRB. Values with different letters (a, b, c, d) are significantly
different from each other (P < 0.05).
|
|
Insulin has been previously demonstrated to be a dominant negative
modulator of IGFBP-1 secretion (26, 48). In the present study,
incubation of cells with a maximally effective insulin concentration
(100 nM) resulted in a 55% reduction in IGFBP-1 mRNA expression,
compared with cells treated with IL-1
alone (Fig.
4). Again, the insulin- and IL-induced
changes in IGFBP-1 protein secretion were comparable to those observed
for mRNA expression (Fig. 4, inset).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Ability of insulin to suppress IL-1 -induced increases in IGFBP-1.
Cells were incubated for 18 h in the presence of insulin (100 nM)
and/or IL-1 (100 ng/ml). Values are means ± SD;
n = 3 plates for each time point.
* P < 0.05 compared with
control values.
+ P < 0.05 compared with cells treated with IL-1 alone. Bar graph
represents IGFBP-1 mRNA abundance quantitated using a
PhosphorImager. Inset:
representative Western blot of IGFBP-1 protein in the medium.
|
|
To determine whether the stimulating effect of IL-1, TNF-
, and IL-6
on IGFBP-1 production was additive or synergistic, Hep G2 cells were
incubated with a relatively low concentration (3 ng/ml) of each
cytokine separately or in combination (Fig.
5). Similar to data presented earlier, both
IGFBP-1 protein and mRNA expression were increased by each cytokine
individually and to a greater extent in those cells treated with
IL-1
. Addition of all three cytokines together resulted in an
additive, or possibly synergistic, effect on the production of IGFBP-1
(Fig. 5, top). The cytokine
combination also enhanced IGFBP-1 mRNA expression, and this effect
appeared to be additive (Fig. 5,
bottom).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of cytokines, individually or in combination, on IGFBP-1
protein in the medium (top) and
cellular mRNA abundance (bottom).
Cells were incubated with 3 ng/ml of TNF- , IL-6, or IL-1 or with
a combination of all three cytokines (3 ng/ml each). Values are means ± SD of 3 plates of cells. Values with different letters (a, b, c,
d) are significantly different (P < 0.05) from each other by ANOVA.
|
|
Oxidative stress-induced changes in IGFBP-1.
t-Butyl-H2O2
increased the medium IGFBP-1 concentration at both 100 and 300 µM
(137 and 180%, respectively; Fig. 6,
top). A qualitatively similar,
albeit quantitatively smaller, increase in IGFBP-1 was observed at the
highest dose of
H2O2
(73%). The in vitro generation of
H2O2
by addition of xanthine and xanthine oxidase also resulted in a small
(68%) but statistically significant increase in the IGFBP-1
concentration (Fig. 6, top). IGFBP-1
secretion was also stimulated by the NO donors SNAP and SNP (Fig. 5,
bottom). Higher doses of exogenous
ROS were not used in this study because preliminary data indicated that
they caused cell death, as evidenced by the increased release of LDH
into the medium. Endogenous generation of peroxynitrite produced by the
decomposition of SIN-1 increased IGFBP-1 levels (45 ± 6 vs. 94 ± 12 ng/ml), and the increment was comparable to that seen
in response to other ROS described above. Although incubation of cells
with
t-butyl-H2O2
(300 µM) or SNAP (0.5 µM) stimulated IGFBP-1 production, no
significant increase in IGFBP-1 mRNA was detected at the 18-h
time point (data not shown).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
Concentration of IGFBP-1 in medium from Hep G2 cells incubated with
various oxidative stressors. Top:
cells were incubated with H2O2 (100 and 300 µM), t-butyl-H2O2
(tb-H2O2; 100 and 300 µM), and xanthine (500 µM) + xanthine oxidase (X/XO; 10 mU/ml).
Bottom: cells incubated with various
concentrations (0.1, 0.5, or 1.0 mM) of
S-nitroso-N-acetylpenicillamine
(SNAP) or sodium nitroprusside (SNP). Vehicle for each drug was culture
medium, except for SNAP, which was dissolved in DMSO. DMSO
concentrations as high as 1% did not alter basal IGFBP-1 secretion.
All cells were incubated for ~18 h. Values are means ± SE;
n = 5-6 sets of duplicate wells.
* P < 0.05 compared with
control value.
|
|
Simultaneous treatment of Hep G2 cells with
H2O2
and IL-1
increased the IGFBP-1 concentration (Fig.
7). However, the increment in IGFBP-1
induced by this combination was not different from that seen in cells
treated with cytokine alone. Hence, the ability of
H2O2
and IL-1
to increase IGFBP-1 appears to be neither additive nor
synergistic.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 7.
Combined effect of
H2O2
and IL-1 on IGFBP-1 production by Hep G2 cells. Agents were added to
cells at the doses (in µM) indicated. All cells were incubated for
~18 h. Values are means ± SE; n = 3-4 sets of duplicate wells.
* P < 0.05 compared with
values from cells receiving no addition and those receiving only
H2O2.
+ P < 0.05 compared with control value only.
|
|
The increase in IGFBP-1 does not appear to be a generalized stress
response of Hep G2 cells, however, because the IGFBP-1 secretion was
not increased by arsenite (100 µM) or endotoxin (1 µM) (data not shown).
Role of ROS, eicosanoids, and NF-
B on
cytokine-induced changes in IGFBP-1.
Table 1 illustrates that
preincubation of hepatoma cells with either ascorbic acid,
-tocopherol, or NAC did not significantly alter either basal or
IL-1
-stimulated increases in IGFBP-1. Similarly, neither these
oxygen free radical scavengers nor DMSO attenuated or prevented the
increment in IGFBP-1 induced by either TNF-
or IL-6 (data not
shown). Likewise, incubation of cells with various inhibitors of NO
synthesis also failed to alter IL-1
- or TNF-
-induced increases in
IGFBP-1 (Table 1 and data not shown). Finally, incubation with either a
cyclooxygenase (indomethacin) or lipoxygenase (NDGA) inhibitor failed
to significantly reduce the increment in IGFBP-1 produced by IL-1
(Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of reactive oxygen species scavengers and nitric oxide synthase
and eicosanoid inhibitors on IL-1 -induced increases in
IGFBP-1 concentration
|
|
Cytokine-induced activation of NF-
B is known to increase the
expression of a wide range of genes. When cells were incubated with
PDTC, a inhibitor of NF-
B activation, the IL-1
-induced increase
in IGFBP-1 protein secretion and mRNA expression was significantly
reduced by 53 and 30%, respectively (Fig.
8). However, preincubation of Hep G2 cells
with a cell-permeable inhibitor of NF-
B nuclear translocation (SN50)
failed to significantly alter either the basal synthesis of IGFBP-1 or
the ability of IL-1
to stimulate IGFBP-1 synthesis (data not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 8.
Inhibition of nuclear factor- B activation by
pyrrolidinedithiocarbamate (PDTC) on IGFBP-1 protein and mRNA
expression. Cells were incubated overnight with no additions (control,
Ctrl), IL-1 (50 ng/ml), or both IL-1 and PDTC (100 µM). Medium
and cells were collected at ~18 h after addition. Values are means ± SD of 3 sets of individual plates. Values with different letters
are significantly (P < 0.05)
different from each other by ANOVA. Medium IGFBP-1 concentration in
this study was higher than that reported in the first series of
experiments because IGFBP-1 and mRNA were determined in 100-mm plates,
as opposed to 24-well plates.
|
|
 |
DISCUSSION |
There is now considerable evidence implicating cytokines in the control
of various components of the growth hormone-IGF axis, particularly on
the regulation of IGFBP-1. It has been previously demonstrated that in
vivo administration of IL-1
, IL-6, or TNF-
markedly increases
circulating levels of IGFBP-1 (9, 13, 41). Moreover, pretreating
animals with a specific IL-1 receptor antagonist has been shown to
completely prevent the increase in IGFBP-1 in blood and liver produced
by bacterial infection (22). Neutralizing antibodies to TNF-
have
also been shown to partially attenuate the increased production of
IGFBP-1 induced by either endotoxin or peritonitis (9, 11). Hence, not
only are cytokines capable of stimulating IGFBP-1 secretion but the
above-mentioned inhibitor studies suggest that they are critical
components in mediating the increase observed during various
inflammatory conditions. The significance of changes in IGFBP-1 during
inflammation as well as the alterations observed during more
physiological adaptations such as fasting and development remain to be
elucidated. However, numerous in vitro studies have demonstrated the
ability of IGFBP-1 to inhibit IGF-I-mediated responses (24).
We have previously demonstrated, using Western blot analysis, that
proinflammatory cytokines can increase the relative concentration of
IGFBP-1 in conditioned medium from Hep G2 cells (41). This observation
has been confirmed and extended by the results of the present study, in
which the cytokine-induced increases in IGFBP-1 have been quantitated
by a specific IRMA. On the basis of IGFBP-1 protein secretion, Hep G2
cells have approximately the same sensitivity to the stimulatory
effects of IL-1, IL-6, and TNF-
(ED50 ~10 ng/ml). However, the
ability of IL-1 (either
- or
-isoform) to maximally stimulate
IGFBP-1 production was greater than for either IL-6 or TNF-
.
Cytokine-induced increases were seen at a nominal concentration of
~1-3 ng/ml, levels that may be present either in the blood or
the local hepatic environment during inflammation (8, 15). Moreover, a
low dose of all three cytokines together appeared to have an additive
or synergistic effect on IGFBP-1 production.
This study clearly indicates that these cytokines also produce a
corresponding increase in IGFBP-1 mRNA abundance. The magnitude of the
cytokine-induced increase in IGFBP-1 mRNA was comparable to that seen
in hepatocytes and rat hepatoma cells incubated with dexamethasone (26,
37). The IL-1-induced increase in IGFBP-1 expression appears to be
regulated at the transcriptional level because cytokine treatment did
not significantly alter IGFBP-1 mRNA stability. Our estimate of IGFBP-1
mRNA half-life in Hep G2 cells under basal conditions is in agreement
with that reported for rat hepatoma cells (36). The present findings
are the first to indicate that IGFBP-1 mRNA is transcriptionally
regulated by IL-1 in hepatic cells. This is the same mechanism by which
insulin and glucocorticoids control IGFBP-1 synthesis (36, 48).
Similarly, the time course for the IL-1
-induced increase in IGFBP-1
mRNA was comparable to that previously observed in cells incubated with
dexamethasone (36), with the half-maximal increase occurring at ~5 h.
Moreover, insulin appears to partially suppress both the IL- and
dexamethasone-induced increase in IGFBP-1 synthesis (26).
The half-life of IGFBP-1 mRNA deduced by DRB treatment and Northern
blotting is consistent with the finding that IGFBP-1 protein increases
in the medium of Hep G2 cells during the first 10 h of DRB treatment
but that additional accumulation is negligible after 15 h when more
than 93% of the IGFBP-1 mRNA has been lost from the cells. DRB
significantly inhibited IGFBP-1 mRNA and protein accumulation
in both control cells and those treated with IL-1
. DRB was chosen as
the transcription inhibitor for these experiments after a preliminary
study demonstrated that another transcription inhibitor, actinomycin D,
paradoxically stimulated the synthesis and secretion of IGFBP-1 and
increased IGFBP-1 mRNA at time points after 12 h. This response to
actinomycin is similar to the superinduction of NF-
B that has been
previously observed in epithelial cells treated with actinomycin D (34)
or the superinduction of IGFBP-1 mRNA in Hep G2 cells treated with
cyclohexamide (35).
A portion of the hepatic response to cytokines is mediated via the
production of ROS, including reactive intermediates of oxygen (i.e.,
H2O2)
and nitrogen (i.e., NO). Hence, the ability of cytokines to increase
IGFBP-1 synthesis may be regulated by the secondary
production of one or more ROS. To determine whether IGFBP-1 production
by Hep G2 cells could be induced by reactive oxygen intermediates
(ROI), cells were incubated with either
H2O2, the more stable
t-butyl-H2O2,
or the combination of xanthine plus xanthine oxidase, which generates
ROI endogenously. This appears to be the first report that ROI are
capable of stimulating IGFBP-1 protein release in vitro. In addition,
reactive NO donors and peroxynitrite were also able to enhance IGFBP-1
production to a comparable extent. It is noteworthy, however, that the
increase in IGFBP-1 production induced by ROS was considerably smaller than that observed in response to cytokine stimulation (2-fold vs. 8- to 10-fold). Finally, no synergistic or additive effect was detected in
cells incubated with both ROS and IL-1
, suggesting that ROS and
IL-1
enhanced IGFBP-1 production via a similar mechanism. In
contrast to the response observed with cytokines, we did not detect an
increase in IGFBP-1 mRNA in cells incubated with ROS. Although this
lack of response has not been further examined, it is possible that ROS
did increase IGFBP-1 mRNA but that the increase was too small to
reliably detect or occurred relatively early after the addition of ROS
and was transient in nature.
Although ROS were able to stimulate IGFBP-1 release, their role in
mediating cytokine-induced changes in Hep G2 cells appears minimal. Our
data indicate that various antioxidants, free radical scavengers, and
inhibitors of inducible and/or constitutive NO synthase failed
to prevent or attenuate the release of IGFBP-1 in response to
cytokines. Similar doses of these inhibitors have been previously
reported to prevent other cytokine-induced responses (as referenced in
MATERIALS AND METHODS), and,
therefore, the failure of these agents to effectively inhibit increases
in IGFBP-1 does not appear related to the use of suboptimal doses.
Although endogenously produced ROS did not appear to mediate
cytokine-induced increases in IGFBP-1 under this particular
experimental paradigm, this does not exclude the possibility that ROS
are important regulators of IGFBP-1 synthesis in other cell types or
under different conditions.
Cytokines are known to stimulate arachidonic acid metabolism, and the
eicosanoids generated appear capable of mediating gene expression (19).
Enzymatic oxidation of arachidonic acid via the cyclooxygenase and
lipoxygenase pathways leads to the synthesis of various prostaglandins,
thromboxanes, and leukotrienes. However, pretreatment of Hep G2 cells
with indomethacin, which selectively inhibits the cyclooxygenase
pathway, or NDGA, which preferentially inhibits the lipoxygenase
pathway at the dose used, had no detectable effect on the
cytokine-induced increase in IGFBP-1.
Transcription factors, such as NF-
B and activator protein-1, are
important in upregulating the expression of specific genes in response
to extracellular signals, including cytokines and ROS (1). The
inducible expression of many of these genes represents a central
cellular response to stress, injury, and inflammation. In the present
study, PDTC significantly attenuated the IL-induced increase in IGFBP-1
mRNA and protein. Because the dithiocarbamates are both inhibitors of
NF-
B activity and potent antioxidants, the exact mechanism for the
PDTC inhibition of the cytokine-induced increase in IGFBP-1 remains
unclear. However, nuclear translocation of NF-
B does not appear
necessary for upregulation of IGFBP-1 synthesis. Preincubation of cells
with SN50, a cell-permeable peptide containing the p50 NF-
B subunit
NLS, failed to block the increase in IGFBP-1 induced by IL-1
. This
peptide has previously been shown to maximally inhibit NF-
B
translocation at the concentration and in the time frame used in the
present study, whereas peptides with a mutated NLS had no effect (28).
These findings are consistent with the ability of PDTC to inhibit
activation of an IL-1
receptor-associated protein kinase and thus
one of the earliest steps in IL-1 signaling after ligand binding to its
receptor (46). Therefore, our data suggest that IL-1
increases
IGFBP-1 synthesis through a PDTC-sensitive, but NF-
B-independent,
pathway, which has been previously hypothesized to mediate other
effects of IL-1
(5).
In summary, our data indicate that in human hepatoma cells IL-1, IL-6,
and TNF-
produce dose-dependent increases in IGFBP-1 protein release
and IGFBP-I mRNA expression and that the increased rate of IGFBP-1
synthesis is due primarily to stimulation of transcription. Moreover,
although ROS are capable of enhancing IGFBP-1 release, cytokine-induced
increases in IGFBP-1 release are not mediated by endogenously produced
ROS and also appear to be independent of eicosanoids. In contrast, part
of the IL-induced increase is mediated via a PDTC-sensitive pathway.
 |
ACKNOWLEDGEMENTS |
We thank the Biological Resources Branch of the Biological Response
Modifiers Program, Division of Cancer Treatment, National Cancer
Institute, for providing the recombinant human IL-1
and -
. We
thank Amgen, Inc., for the generous gift of TNF-
.
 |
FOOTNOTES |
This work was supported in part by National Institutes of Health Grants
GM-38032 and AA-11290.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. H. Lang,
Cell. Molec. Physiology (H166), Pennsylvania State College of Medicine,
500 University Dr., Hershey, PA 17033-0850 (E-mail:
clang{at}psu.edu).
Received 14 July 1998; accepted in final form 3 December 1998.
 |
REFERENCES |
1.
Barnes, H.,
and
M. Karin.
Nuclear factor-
B
a pivotal transcription factor in chronic inflammatory diseases.
N. Engl. J. Med.
336:
1066-1071,
1997[Free Full Text].
2.
Bautista, A. P.,
K. Meszaros,
J. Bojta,
and
J. J. Spitzer.
Superoxide anion generation in the liver during the early stage of endotoxemia in rats.
J. Leukoc. Biol.
48:
123-128,
1990[Abstract].
3.
Bautista, A. P.,
and
J. J. Spitzer.
Superoxide anion generation by in situ perfused rat liver: effect of in vivo endotoxin.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G907-G912,
1990[Abstract/Free Full Text].
4.
Bereket, A.,
C. H. Lang,
S. L. Blethen,
M. C. Gelato,
J. Fan,
R. A. Frost,
and
T. A. Wilson.
Effect of insulin on the alterations of growth hormone-IGF axis in children with new onset insulin dependent diabetes mellitus.
J. Clin. Endocrinol. Metab.
80:
1312-1317,
1995[Abstract].
5.
Bergmann, M.,
L. Hart,
M. Lindsay,
P. J. Barnes,
and
R. Newton.
I
B
degradation and nuclear factor-
B DNA binding are insufficient for interleukin-1
and tumor necrosis factor-
-induced
B-dependent transcription. Requirement for an additional activation pathway.
J. Biol. Chem.
273:
6607-6610,
1998[Abstract/Free Full Text].
6.
Cichy, S. B.,
S. Uddin,
A. Danilkovich,
S. Guo,
A. Klippel,
and
T. G. Unterman.
Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence.
J. Biol. Chem.
273:
6482-6487,
1998[Abstract/Free Full Text].
7.
Delany, A. M,
and
E. Canalis.
Transcriptional repression of insulin-like growth factor I by glucocorticoids in rat bone cells.
Endocrinology
136:
4776-4781,
1995[Abstract].
8.
Ertel, W.,
M. H. Morrison,
P. Wang,
Z. F. Ba,
A. Ayala,
and
I. H. Chaudry.
The complex pattern of cytokines in sepsis. Association between prostaglandins, cachectin, and interleukins.
Ann. Surg.
214:
141-148,
1991[Medline].
9.
Fan, J.,
G. J. Bagby,
M. C. Gelato,
and
C. H. Lang.
Regulation of insulin-like growth factor I content and IGF-binding proteins by tumor necrosis factor.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R1204-R1212,
1995[Abstract/Free Full Text].
10.
Fan, J.,
D. Char,
A. J. Kolasa,
W. Pan,
S. R. Maitra,
C. S. Patlak,
Z. Spolarics,
M. C. Gelato,
and
C. H. Lang.
Alterations in hepatic production and peripheral clearance of IGF-I after endotoxin.
Am. J. Physiol.
269 (Endocrinol. Metab. 32):
E33-E42,
1995[Abstract/Free Full Text].
11.
Fan, J.,
Y. H. Li,
G. J. Bagby,
and
C. H. Lang.
Modulation of inflammation-induced changes in insulin-like growth factor (IGF)-I and IGF binding protein-1 by anti-TNF antibody.
Shock
4:
21-26,
1995[Medline].
12.
Fan, J.,
P. E. Molina,
M. C. Gelato,
and
C. H. Lang.
Differential tissue regulation of insulin-like growth factor I content and binding proteins after endotoxin.
Endocrinology
134:
1685-1692,
1994[Abstract].
13.
Fan, J.,
M. M. Wojnar,
M. Theodorakis,
and
C. H. Lang.
Regulation of insulin-like growth factor (IGF)-I mRNA and peptide, and IGF binding proteins by interleukin-1.
Am. J. Physiol.
270 (Regulatory Integrative Comp. Physiol. 39):
R621-R629,
1996[Abstract/Free Full Text].
14.
Geller, D. A.,
A. K. Nussler,
M. DiSilvio,
C. J. Lowenstein,
R. A. Shapiro,
S. C. Wang,
R. L. Simmons,
and
T. R. Billiar.
Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA
90:
522-526,
1993[Abstract].
15.
Givalois, L.,
J. Dornand,
M. Mekaouche,
M. D. Solier,
A. F. Bristow,
G. Ixart,
P. Siaud,
I. Assenmacher,
and
G. Barbanel.
Temporal cascade of plasma level surges in ACTH, corticosterone, and cytokines in endotoxin-challenged rats.
Am. J. Physiol.
267 (Regulatory Integrative Comp. Physiol. 36):
R164-R170,
1994[Abstract/Free Full Text].
16.
Ikeda, K.,
S. Kubo,
K. Hirohashi,
H. Kinoshita,
K. Kaneda,
N. Kawada,
E. F. Kato,
and
M. Inoue.
Mechanisms that regulates nitric oxide production by lipopolysaccharide-stimulated rat Kupffer cells.
Physiol. Chem. Phys. Med. NMR
28:
239-243,
1996[Medline].
17.
Ito, A.,
T. Takii,
T. Soji,
and
K. Onozaki.
Endotoxin-induced upregulation of type I interleukin-1 receptor mRNA expression hepatocytes of mice: role of cytokines.
J. Interferon Cytokine Res.
17:
55-61,
1997[Medline].
18.
Jaeschke, H.,
and
C. W. Smith.
Mechanisms of neutrophil-induced parenchymal cell injury.
J. Leukoc. Biol.
61:
647-653,
1997[Abstract].
19.
Jurivich, D. A.,
L. Sistonen,
K. D. Sarge,
and
R. I. Morimoto.
Arachidonate is a potent modulator of heat shock gene transcription.
Proc. Natl. Acad. Sci. USA
91:
2280-2286,
1991[Abstract].
20.
Kim, Y.-M.,
M. E. de Vera,
S. C. Watkins,
and
T. R. Billiar.
Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-
-induced apoptosis by inducing heat shock protein 70 expression.
J. Biol. Chem.
272:
1402-1411,
1997[Abstract/Free Full Text].
21.
Kurose, I.,
S. Miura,
H. Higuchi,
N. Watanabe,
Y. Kamegaya,
M. Takaishi,
K. Tomita,
D. Fukumura,
S. Kato,
and
H. Ishii.
Increased nitric oxide synthase activity as a cause of mitochondrial dysfunction in rat hepatocytes: roles for tumor necrosis factor
.
Hepatology
24:
1185-1192,
1996[Medline].
22.
Lang, C. H.,
J. Fan,
R. Cooney,
and
T. C. Vary.
Interleukin-1 receptor antagonist attenuates sepsis-induced alterations in the insulin-like growth factor system and protein synthesis.
Am. J. Physiol.
270 (Endocrinol. Metab. 33):
E430-E437,
1996[Abstract/Free Full Text].
23.
Lee, P. D. K.,
L. S. Abdel-Maguid,
and
M. B. Snuggs.
Role of protein kinase C in regulation of insulin-like growth factor binding protein-1 production by HepG2 cells.
J. Clin. Endocrinol. Metab.
75:
459-464,
1992[Abstract].
24.
Lee, P. D. K.,
C. Conover,
and
D. R. Powell.
Regulation and function of insulin-like growth factor-binding protein-1. Proc.
Soc. Exp. Biol. Med.
204:
4-29,
1993[Abstract].
25.
Lee, P. D. K.,
M. D. Jensen,
G. D. Divertie,
V. J. Heiling,
H. H. Katz,
and
C. A. Conover.
Insulin-like growth factor binding protein-1 response to insulin during suppression of endogenous insulin secretion.
Metabolism
42:
409-414,
1993[Medline].
26.
Lewitt, M. S.,
H. Saunders,
and
R. C. Baxter.
Interaction of insulin, glucocorticoids, and protein kinase C in the regulation of insulin-like growth factor-binding protein-1 production by H4IIE rat hepatoma cells.
J. Cell. Physiol.
166:
121-129,
1996[Medline].
27.
Li, Y. H.,
J. Fan,
and
C. H. Lang.
Role of glucocorticoids in mediating endotoxin-induced changes in IGF-I and IGF binding protein-1.
Am. J. Physiol.
272 (Regulatory Integrative Comp. Physiol. 41):
R1970-R1977,
1997.
28.
Lin, Y. Z.,
S. Y. Yao,
R. A. Veach,
T. R. Torgerson,
and
J. Hawiger.
Inhibition of nuclear translocation of transcription factor NF-
B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.
J. Biol. Chem.
270:
14255-14258,
1997[Abstract/Free Full Text].
29.
Long, S. D.,
and
P. H. Pekala.
Regulation of GLUT 4 gene expression by arachidonic acid.
J. Biol. Chem.
271:
1138-1144,
1996[Abstract/Free Full Text].
30.
Mackiewicz, A.,
T. Speroff,
M. K. Ganapathi,
and
I. Kushner.
Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines.
J. Immunol.
146:
3032-3037,
1991[Abstract/Free Full Text].
31.
McCusker, R. H.,
and
D. R. Clemmons.
The insulin-like growth factor binding proteins: structure and biological functions.
In: The Insulin-Like Growth Factors: Structure and Biological Functions, edited by P. N. Schofield. New York: Oxford University Press, 1992, p. 110-150.
32.
Motoyama, S.,
Y. Minamiya,
S. Saito,
R. Saito,
I. Matsuzaki,
S. Abo,
H. Inaba,
K. Enomoto,
and
M. Kitamura.
Hydrogen peroxide derived from hepatocytes induces sinusoidal endothelial cell apoptosis in perfused hypoxic rat liver.
Gastroenterology
114:
153-163,
1998[Medline].
33.
Musonda, C. A.,
and
J. K. Chipman.
Quercetin inhibits hydrogen peroxide-induced NF-
B DNA binding activity and DNA damage in HepG2 cells.
Carcinogenesis
19:
1583-1589,
1998[Abstract].
34.
Newton, R.,
I. M. Adcock,
and
P. J. Barnes.
Superinduction of NF-
B by actinomycin D and cycloheximide in epithelial cells.
Biochem. Biophys. Res. Commun.
218:
518-523,
1996[Medline].
35.
Ooi, G. T.,
D. R. Brown,
D. S. Suh,
L. Y. Tseng,
and
M. M. Rechler.
Cycloheximide stabilizes insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and inhibits IGFBP-1 transcription in H4-II-E rat hepatoma cells.
J. Biol. Chem.
268:
16664-16672,
1993[Abstract/Free Full Text].
36.
Orlowski, C. C.,
G. T. Ooi,
and
M. M. Rechler.
Dexamethasone stimulates transcription of the insulin-like growth factor-binding protein-1 gene in H4-II-E rat hepatoma cells.
Mol. Endocrinol.
4:
1592-1599,
1990[Abstract].
37.
Pao, C.-I.,
P. K. Farmer,
S. Begovic,
B. C. Villafuerte,
G. Wu,
D. G. Robertson,
and
L. S. Phillips.
Regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein 1 gene transcription by hormones and provision of amino acids in rat hepatocytes.
Mol. Endocrinol.
7:
1561-1568,
1993[Abstract].
38.
Powell, D. R.,
A. Suwanichkul,
M. L. Cubbage,
L. A. DePaolis,
M. B. Snuggs,
and
P. D. K. Lee.
Insulin inhibits transcription of the human gene for insulin-like growth factor-binding protein-1.
J. Biol. Chem.
266:
18868-18876,
1991[Abstract/Free Full Text].
39.
Reddy, S. A. G.,
J. H. Huang,
and
W. S. L. Liao.
Phosphatidylinositol 3-kinase in interleukin-1 signaling.
J. Biol. Chem.
272:
29167-29173,
1997[Abstract/Free Full Text].
40.
Rohrdanz, E.,
and
R. Kahl.
Alterations of antioxidant enzyme expression response to hydrogen peroxide.
Free Radic. Biol. Med.
24:
27-38,
1998[Medline].
41.
Samstein, B.,
M. L. Hoimes,
J. Fan,
R. A. Frost,
M. C. Gelato,
and
C. H. Lang.
IL-6 stimulation of IGFBP-1 production.
Biochem. Biophys. Res. Commun.
228:
611-615,
1996[Medline].
42.
Sanchez, A.,
A. M. Alvarez,
M. Benito,
and
I. Fabregat.
Apoptosis induced by transforming growth factor-
in fetal hepatocyte primary culture. Involvement of reactive oxygen intermediates.
J. Biol. Chem.
271:
7416-7422,
1996[Abstract/Free Full Text].
43.
Stadler, J.,
B. G. Bentz,
B. G. Harbrecht,
M. Di Silvio,
R. D. Curran,
T. R. Billiar,
R. A. Hoffman,
and
R. L. Simmons.
Tumor necrosis factor
inhibits hepatocyte mitochondrial respiration.
Ann. Surg.
216:
539-546,
1992[Medline].
44.
Suwanickul, A.,
S. L. Morris,
and
D. R. Powell.
Identification of an insulin-responsive element in the promoter of the human gene for insulin-like growth binding protein-1.
J. Biol. Chem.
268:
17063-17068,
1993[Abstract/Free Full Text].
45.
Tazuke, S. I.,
N. M. Mazure,
J. Sugawara,
G. Carland,
G. H. Fassen,
L. F. Suen,
J. C. Irwin,
D. R. Powell,
A. J. Gaicca,
and
L. C. Giudice.
Hypoxia stimulates insulin-like growth factor binding protein-1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia.
Proc. Natl. Acad. Sci. USA
95:
10188-10193,
1998[Abstract/Free Full Text].
46.
Tewes, F.,
G. F. Bol,
and
R. Brigelius-Flohe.
Thiol modulation inhibits the interleukin (IL)-1 mediated activation of an IL-1 receptor-associated protein kinase and NF-
B.
Eur. J. Immunol.
27:
3015-3021,
1998.
47.
Ulich, T. R.,
K. Guo,
D. Remick,
J. del Castillo,
and
S. Yin.
Endotoxin-induced cytokine gene expression in vivo. III. IL-6 mRNA and serum protein expression and the in vivo hematologic effects of IL-6.
J. Immunol.
146:
2316-2323,
1991[Abstract/Free Full Text].
48.
Unterman, T. G.,
D. T. Oehler,
L. J. Murphy,
and
R. G. Lacson.
Multihormonal regulation of insulin-like growth factor binding protein-1 in rat H4EII hepatoma cells: the dominant role of insulin.
Endocrinology
128:
2693-2701,
1991[Abstract].
49.
Wang, J. F.,
S. S. Greenberg,
and
J. J. Spitzer.
Chronic alcohol administration stimulates nitric oxide formation in the rat liver with or without pretreatment by lipopolysaccharide.
Alcohol. Clin. Exp. Res.
19:
387-393,
1995[Medline].
Am J Physiol Gastroint Liver Physiol 276(3):G719-G727
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society