Department of Physiology, University of Cambridge, Cambridge CB2 3EG, United Kingdom
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ABSTRACT |
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Tumor necrosis factor- (TNF-
) is an important component of
the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-
on thapsigargin (TG)-induced store-mediated Ca2+ entry (SMCE) in the human
hepatocellular carcinoma cell line HepG2. In these cells, short-term
(10 min) exposure to TNF-
slightly increased SMCE. In contrast,
long-term (12 h) exposure to TNF-
significantly reduced SMCE. This
effect was reversed by coincubation with atrial natriuretic peptide
(ANP), which itself had no effect on SMCE. Cytochalasin D and
latrunculin A, inhibitors of actin polymerization, abolished SMCE.
Long-term exposure of HepG2 cells to TNF-
abolished TG-induced actin
polymerization and membrane association of Ras proteins. When TNF-
was added in combination with ANP, these effects were reduced. These
findings suggest that in HepG2 cells, TNF-
inhibits SMCE by
affecting reorganization of the actin cytoskeleton, probably by
interfering with the activation of Ras proteins, and that ANP protects
against these inhibitory effects of TNF-
.
calcium influx; actin cytoskeleton; Ras proteins; atrial natriuretic peptide
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INTRODUCTION |
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SEVERAL STUDIES HAVE
REPORTED that the pleiotropic cytokine tumor necrosis factor-
(TNF-
) induces several, often opposing, cellular effects in a
cell-specific manner, including its capacity to promote cell survival
under some circumstances while inducing apoptosis in others
(8). The recent discovery that TNF-
recognizes two cell
surface receptors that could mediate different cellular effects sheds
light on the understanding of the biological activity of TNF-
(8). In liver cells, TNF-
has been suggested to have a
dual effect. It plays an important role in the early signaling pathways
leading to regeneration (21). However, long-term exposure to TNF-
is also known to induce apoptosis in hepatocytes
(15). Several studies have suggested a close relationship
between TNF-
and atrial natriuretic peptide (ANP) in different
tissues (34, 39). Vollmar et al. (43)
proposed an autocrine model for ANP in liver cells. Since the discovery
of ANP, several biological activities have been reported, most notably,
renal and cardiovascular effects (5). In addition, there
is increasing evidence for actions affecting liver functions. A
cytoprotective effect of ANP in reperfusion injury and damage by oxygen
radicals has been described (2, 27, 34).
A recent study reported that short-term exposure to TNF-
decreased thapsigargin (TG)-induced Ca2+ entry in thyroid
FRTL-5 cells, although the mechanism of action of TNF-
remains
unclear (42). Store-mediated Ca2+ entry (SMCE)
is a mechanism present in many cell types, including hepatocytes
(18). Several hypotheses consider both direct and indirect
coupling mechanisms for the activation of SMCE (25). Recently, a secretion-like coupling model has been proposed in several
cell types, which involves a physical but reversible interaction between the endoplasmic reticulum and the plasma membrane (26, 30, 47). This mechanism may require translocation of portions of
the endoplasmic reticulum toward the plasma membrane and mechanical support provided by the actin cytoskeleton (28, 31). In
support of this model, small GTP-binding proteins, which
modulate intracellular transport through the reorganization of the
actin cytoskeleton and the actin cytoskeleton itself, have been
suggested to play a regulatory role in the activation of SMCE (3,
7, 32). Here we report for the first time that TNF-
-induced
inhibition of SMCE might be mediated by inhibition of the membrane
association of Ras proteins and actin polymerization in HepG2 cells. In
addition, we demonstrate that ANP has a protective effect against this
TNF-
-evoked inhibition.
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MATERIALS AND METHODS |
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Materials.
Fura 2-acetoxymethyl ester (fura 2-AM) was purchased from Texas
Fluorescence (Austin, TX). Paraformaldehyde, Nonidet P-40, Hoechst
33342, TNF-, FITC-labeled phalloidin, fetal calf serum, and TG were
from Sigma (Poole, UK). Cytochalasin D (Cyt D) was from Calbiochem
(Nottingham, UK). 1,4-Di-azobicyclo-(2.2.2.)octane (DABCO) was from
GIBCO BRL (Paisley, UK). Pan-Ras (Ab-3) monoclonal antibody was
from Oncogene Science (Cambridge, MA). ANP and
-ANP polyclonal
antibody were from Peptide Institute (Osaka, Japan). Horseradish
peroxidase-conjugated ovine anti-mouse IgG antibody (NA931),
biotinylated anti-rabbit IgG antibody, and Cy3-Streptavidin were from
Amersham (Amersham, UK). Dimethyl
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA)-AM and latrunculin A (Lat A) were from Molecular Probes
(Leiden, The Netherlands). All other reagents were of analytical grade.
Cell culture. The human hepatoblastoma cell line HepG2 was obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) and grown at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing high glucose levels. Culture medium was supplemented with 10% heat-inactivated fetal bovine serum (BSA).
Measurement of intracellular free Ca2+ concentration. HepG2 cells were incubated at room temperature (20°C) with 2 µM fura 2-AM for 30 min. For coloading with dimethyl BAPTA, cells were incubated with 10 µM dimethyl BAPTA-AM for 30 min at room temperature. Cells were then collected by centrifugation at 170 g for 5 min and resuspended in HEPES-buffered saline (HBS) containing (in mM) 145 NaCl, 10 HEPES, 10 D-glucose, 2.5 probenecid, 5 KCl, and 1 MgSO4, pH 7.45, and supplemented with 0.1% wt/vol BSA. Fluorescence was recorded from 1.5-ml aliquots of magnetically stirred cell suspension (2 × 106 cells/ml) at 37°C with the use of a spectrophotometer (Cairn Research, Sittingbourne, UK) with excitation wavelengths of 340 and 380 nm and emission at 500 nm. Changes in intracellular free Ca2+ concentration ([Ca2+]i) were monitored with the fura 2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al. (11). Mn2+ influx was monitored as a quenching of fura 2 fluorescence at the isoemissive wavelength of 360 nm, presented on an arbitrary linear scale (36).
Determination of Ca2+ entry. Ca2+ influx in TG-induced store-depleted cells was estimated as described previously (12). Briefly, Ca2+ entry was estimated by using the integral against time of the rise in [Ca2+]i above basal level (the level before the addition of CaCl2) for 1.5 min after CaCl2 was added. When hepatocytes were preincubated with inhibitors, Ca2+ entry was corrected by subtraction of the rise in [Ca2+]i due to leakage of the indicator.
Measurement of F-actin content. The F-actin content of resting and activated HepG2 cells was determined as previously described (32). Briefly, HepG2 cells (106 cells/ml) were activated in HBS. Samples of cell suspension (200 µl) were transferred to 200-µl ice-cold 3% (w/vol) formaldehyde in phosphate-buffered saline (PBS) for 10 min. Fixed cells were permeabilized by incubation for 10 min with 0.025% (vol/vol) Nonidet P-40 detergent dissolved in PBS and were then incubated for 30 min with FITC-labeled phalloidin (1 µM) in PBS supplemented with 0.5% (wt/vol) BSA. After incubation, cells were collected by centrifugation in a Micro-Centaur centrifuge (MSE Scientific Instruments, Crawley, UK) for 90 s at 3,000 g and then resuspended in PBS. Staining of cells was measured with a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT). Samples were excited at 496 nm, and emission was at 516 nm. For confocal microscopy of the actin cytoskeleton, HepG2 cells were stained according to the same protocol. Cells were then mounted in poly-L-lysine-coated coverslips and visualized with a Leica TCS4D confocal microscope.
Immunocytochemistry.
Cells were seeded onto poly-L-lysine-coated glass
coverslips in 4- or 24-well plates and grown in DMEM with 10% fetal
calf serum. Monolayers were washed once or twice in PBS, fixed for 2 min in cold methanol (20°C), washed twice with PBS containing 0.1%
BSA (PBS/BSA) for 5 min each, and blocked with PBS containing 3% BSA,
20 mM poly-L-lysine, and 5% donkey serum. Cells were then incubated for 2 h with rabbit polyclonal
-ANP antibody diluted 1:200 in PBS containing 3% BSA and 20 mM poly-L-lysine, pH
7.4. After three washes in PBS/BSA, cells were incubated for 1 h
at room temperature with biotinylated anti-rabbit IgG secondary
antibody diluted 1:200. The cells were washed three times, as before,
and incubated for 1 h with Cy3-Streptavidin diluted 1:5,000 and
Hoechst 33342 diluted 1:5,000. Cells were washed again three times in PBS/BSA and once in PBS and then mounted in glycerol-Tris-buffered saline (9:1) containing an anti-fading agent (2.5% DABCO).
Subcellular fractionation. Cell fractionation was carried out according to a procedure published previously (32). Briefly, cells were pelleted in a microcentrifuge, and the pellets were quickly resuspended in 0.5 ml of ice-cold Tris · HCl buffer containing 10 mM Tris · HCl (pH 7.2), 158 mM NaCl, 1 mM EGTA, 50 µg/ml leupeptin, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. The suspensions were sonicated, and intact cells were removed by centrifugation at 1,500 g. The whole cell lysate was centrifuged at 100,000 g at 4°C for 30 min to obtain membrane and cytosolic fractions. Membranes were washed with PBS containing 1 mM Na3VO4 at 4°C and resuspended in Tris · HCl buffer containing 10 mM Tris · HCl (pH 7.2), 158 mM NaCl, 1 mM EGTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 50 µg/ml leupeptin, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. Lysates were centrifuged at 16,000 g for 5 min to remove insoluble substances. One-dimensional SDS electrophoresis was performed with 10% polyacrylamide minigels, and separated proteins were electrophoretically transferred, for 2 h at 0.8 mA/cm2, in a semi-dry blotter (Hoefer Scientific, Newcastle, UK) onto nitrocellulose for subsequent probing. Blots were analyzed by Western blotting with pan-Ras (Ab-3) monoclonal antibody diluted 1:300 in Tris-buffered saline with 0.1% Tween 20 (TBST). To detect the primary antibody, we incubated blots with horseradish peroxidase-conjugated anti-mouse IgG antibody diluted 1:10,000 in TBST and then exposed them to enhanced chemiluminescence reagents for 1 min. Blots were then exposed to preflashed photographic film.
Statistical analysis. Analysis of statistical significance was performed using Student's paired t-test. The significance level was P < 0.05.
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RESULTS |
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Regulation of ANP immunoreactivity by TNF-.
HepG2 cells were treated with TNF-
(100 ng/ml) or the vehicle for
12 h and then incubated with anti-
-ANP antibody to identify
-ANP and also with Hoechst 33342 to identify the nucleus of the cells. Treatment of HepG2 cells for 12 h with TNF-
(Fig.
1A, ii) resulted in an
increase in ANP immunoreactivity compared with its relative control
(vehicle-treated cells; Fig. 1A, i; n = 5). Staining of cells with Hoechst 33342 confirmed that a similar density
of cells appears (Fig. 1B, i and ii;
n = 5).
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TG activates store-mediated divalent cation entry in HepG2 cells.
In a Ca2+-free medium (100 µM EGTA was added), treatment
of HepG2 cells with 3 µM TG, a specific inhibitor of the
Ca2+-ATPase of internal stores (SERCA; Ref.
41), evoked a prolonged elevation of
[Ca2+]i in HepG2 cells due to release of
Ca2+ from intracellular pools (Fig.
2A). Subsequent addition of
Ca2+ (5 mM) to the external medium induced a sustained
increase in [Ca2+]i, indicative of SMCE (Fig.
2A), a phenomenon that was not observed in cells not treated
with TG (Fig. 2A).
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Effect of TNF-, ANP, or a combination of both on SMCE in HepG2
cells.
Treatment of cells for 10 min with 100 ng/ml TNF-
slightly modified
TG-induced Ca2+ entry (Fig.
3; P < 0.05;
n = 8) without having any effect on TG-induced release
of Ca2+ from the stores (Fig. 3A). Similar
results were observed when the cells were treated with 1 µM ANP
(n = 8) or a combination of both agents (Fig. 3;
P < 0.05; n = 8). In contrast,
long-term exposure of HepG2 cells for 12 h to TNF-
(100 ng/ml)
decreased Ca2+ entry by 46 ± 5% compared with
untreated control cells (Fig. 4;
P < 0.01; n = 8) without having any
effect on TG-induced release of Ca2+ from the intracellular
stores (Fig. 4A). In contrast, exposure of cells to ANP (1 µM) for 12 h did not significantly modify either TG-induced
Ca2+ release or Ca2+ influx (data not shown)
but, surprisingly, significantly reversed the effect of TNF-
when
the two agents were added together. ANP reversed the inhibitory effect
of TNF-
on SMCE to 63 ± 6% of control (Fig. 4;
P < 0.05; n = 8).
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Role of the actin cytoskeleton in SMCE in HepG2 cells.
Recent studies have suggested that the actin cytoskeleton plays a key
regulatory role in the activation of SMCE (9, 13, 30). To
investigate the role of the actin cytoskeleton in the activation of
SMCE in HepG2 cells, we used Cyt D and Lat A, two agents that inhibit
actin polymerization by different mechanisms. Stimulation of HepG2
cells with TG in a Ca2+-free medium (100 µM EGTA was
added) markedly increased actin filament content by 60 ± 6% of
control (Table 1; P < 0.001; n = 6). Pretreatment of cells for 1 h with
Cyt D (10 µM), a widely utilized membrane-permeant inhibitor of actin
polymerization that binds to the barbed end of actin filaments
(44), resulted in a reduction of actin filament content of
unstimulated cells to 68.9 ± 1.1% of control (Table 1;
P < 0.01; n = 6). Treatment of HepG2
cells with Cyt D abolished TG-evoked actin polymerization (Table 1;
P < 0.001; n = 6). Similar results
were obtained with Lat A, an agent that inhibits actin polymerization
by binding to actin monomers (38). Treatment of HepG2
cells with Lat A (3 µM) for 1 h at 37°C both reduced the actin
filament content in nonstimulated cells and abolished TG-induced actin
polymerization (Table 1; P < 0.001; n = 6).
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Effect of TNF-, ANP, or a combination of both on TG-induced
actin polymerization in HepG2 cells.
To investigate whether the inhibitory effect of TNF-
on SMCE is
mediated by interference with actin polymerization, we pretreated HepG2
cells with 100 ng/ml TNF-
for 12 h and determined actin filament content using FITC-phalloidin. As shown in Fig.
7A, long-term exposure to
TNF-
abolished TG-stimulated actin filament formation in HepG2 cells
(P < 0.001; n = 5). TNF-
slightly,
but not significantly, reduced the actin filament content of
unstimulated HepG2 cells by 7% when treated for up to 12 h
(P = 0.25; n = 5). Consistent with the
findings reported above, 12 h of exposure to ANP had no
significant effect on either TG-induced actin polymerization (Fig.
7A; P = 0.57; n = 5) or the
actin filament content in unstimulated cells (data not shown).
Interestingly, ANP was able to significantly reverse the inhibitory
effect of TNF-
on TG-induced actin polymerization; the effect was
very similar to that which ANP had on SMCE when added together with
TNF-
(Fig. 7A; P < 0.05;
n = 5).
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Effect of TNF-, ANP, or both on the subcellular localization of
Ras proteins.
Several proteins of the Ras family have been shown to be involved in
actin polymerization. In addition, it has been reported that inhibition
of the membrane association of Ras proteins is an important process for
Ras activation (20). Therefore, we investigated whether
inhibition of SMCE and actin polymerization by TNF-
in HepG2 cells
might be associated with alterations in the subcellular localization of
Ras. As shown in Fig. 8, in resting HepG2
cells, pan-Ras (Ab-3) immunoreactive proteins could be detected in both
the cytosolic and membrane fractions. In agreement with previous
studies (20, 32), after stimulation with TG, Ras was found
to be localized predominantly in the membrane fraction. When cells were
treated for 12 h with 100 ng/ml TNF-
, most of the Ras proteins
were localized in the cytosolic fraction (Fig. 8; n = 3). Exposure of HepG2 cells for 12 h to 1 µM ANP did not alter
the translocation of Ras to the membrane stimulated by TG, but ANP
reversed the effect of TNF-
on Ras association with membranes (Fig.
8; P < 0.01; n = 3). Treatment for up
to 12 h with TNF-
, ANP, or both had no effect on the cellular
distribution of Ras in unstimulated HepG2 cells (data not shown).
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DISCUSSION |
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TNF- is a potent proinflammatory cytokine secreted by different
cells, including hepatocytes (22), in response to several stimuli such as ethanol (22), hepatectomy
(19), or liver diseases (4), inducing a
variety of biochemical or functional responses in hepatic cells
(10).
The results presented in this study indicate that treatment of the
human hepatocellular carcinoma cell line (HepG2) for 12 h with
TNF- resulted in inhibition of SMCE. TNF-
selectively inhibited
TG-induced SMCE without having any effect on the release of
Ca2+ from the intracellular stores, which indicates that
TNF-
did not affect the ability of HepG2 cells to store
Ca2+ in intracellular compartments. The inhibition of
Ca2+ entry by TNF-
was not due to nonspecific effects
such as chelation of Ca2+ or Ca2+-channel
blockage, as demonstrated by the lack of effect of short-term treatment
of HepG2 cells with TNF-
.
A number of studies have shown that TNF- regulates several processes
in HepG2 cells, such as the expression of the transglutaminase gene
(16) and secretion of phospholipase A2
(45), and it also upregulates the expression of
low-density lipoprotein receptors and stimulates hepatic lipid
synthesis and secretion (17). Despite the important
modulatory role of this cytokine in liver function, relatively little
is known about TNF-
signaling from the cell membrane via the
cytoplasm to the genome. Several groups have suggested that cyclic
nucleotides may play a role in modifying TNF-
synthesis (4,
19). Interestingly, we found that 12 h of incubation of
cells with TNF-
increased ANP immunoreactivity, one of the most
potent and naturally occurring agents that regulate the levels of cGMP
(10). Furthermore, a very recent study (14) suggests that ANP may act to decrease expression of TNF-
mRNA during
reperfusion of the rat liver. Also, we have recently reported (33) that ANP was able to partially protect hepatocytes
against TNF-
-induced apoptosis, further suggesting a close
relationship between these two factors in liver injury.
In our experiments, the incubation of HepG2 cells for up to 12 h
with ANP alone did not alter SMCE. However, when cells were treated
with both TNF- and ANP, the inhibition of Ca2+ entry
observed when cells were treated with TNF-
alone was significantly reduced, indicating that ANP has a protective effect against
TNF-
-induced inhibition of SMCE. In agreement with our findings, the
effect of ANP in modulating TNF-
-induced responses has been recently shown in the human renal proximal tubule, where ANP inhibited TNF-
-stimulated nitric oxide production (6).
It remains to be fully elucidated how TNF- might act to regulate
SMCE. Several hypotheses proposed for the activation of SMCE can be
divided into those that consider a direct interaction between proteins
in the intracellular Ca2+ stores and the plasma membrane
and those that suggest the existence of a diffusible messenger
(1, 25, 35). Recent studies have proposed a new model
based on a physical but reversible interaction between the
intracellular Ca2+ stores and the plasma membrane, where
the actin cytoskeleton plays a key regulatory role (26, 30,
47). To determine whether the actin cytoskeleton plays a role in
SMCE in HepG2 cells, we used the inhibitors of actin polymerization:
Cyt D, a fungal metabolite that blocks the formation of actin
microfilaments by preventing monomer addition at the growing end of the
polymer (44), and Lat A, an agent that inhibits actin
polymerization by binding to actin monomers (38).
Treatment for 1 h with 10 µM Cyt D or 3 µM Lat A reduced the
actin filament content of unstimulated HepG2 cells and abolished
TG-evoked actin polymerization. Treatment with both agents resulted in
the loss of a normal cytoskeletal organization. However, Cyt D- or Lat
A-treated HepG2 cells retained their ability to respond to
Ca2+-mobilizing agents such as TG, which indicates that
these treatments did not affect the amount of Ca2+ stored
in intracellular compartments. Our results clearly demonstrate that
treatment of HepG2 cells with Cyt D or Lat A abolishes the Ca2+ entry following store depletion. This is in agreement
with recent reports describing the effect of actin cytoskeleton
disruption in vascular endothelial cells (13), platelets
(32), and astrocytes (9). These results
suggest that actin filament polymerization is essential for the
activation of SMCE in HepG2 liver cells.
Because actin polymerization is required for SMCE in HepG2 cells, we
investigated the effects of TNF- and ANP on actin filament formation
in these cells. Our results show that long-term exposure of cells to
TNF-
inhibited the actin polymerization stimulated by TG in both
control and dimethyl BAPTA-loaded cells, indicating that this event is
independent of the [Ca2+]i. In addition,
TNF-
slightly reduced the actin filament content of unstimulated
cells. In contrast, ANP did not alter either the actin filament content
of resting cells or TG-induced actin polymerization. Consistent with
the results obtained by measuring SMCE, ANP partially reversed
TNF-
-induced inhibition of actin polymerization. As shown for the
Ca2+ entry mechanism, 10 min of preincubation with TNF-
had no significant effect on the actin filament content of resting or
TG-stimulated cells. The parallel between SMCE and actin polymerization
and the effect of the actin polymerization inhibitors on
Ca2+ entry allows us to propose that TNF-
-induced
inhibition of SMCE in HepG2 cells may be mediated by the inhibition of
actin polymerization in these cells. To our knowledge, this is the
first demonstration of the inhibitory effect of TNF-
on SMCE and
actin polymerization and of the reversal of these effects by ANP. These
results are of particular interest in light of the recent observation
that the hormonal elevation of [Ca2+]i and
the resulting activation of specific metabolic pathways in the liver
require actin filament reorganization (46).
Many cell functions, including the maintenance of morphology,
aggregation, motility, smooth-muscle contraction, and membrane ruffling, are regulated by small GTP-binding proteins of the Ras superfamily through the dynamic reorganization of the actin
cytoskeleton (23, 24, 29, 40). We have previously shown
that inhibition of the membrane association of Ras proteins, which has
been reported to be essential for Ras activation (20),
inhibits both actin polymerization and SMCE in human platelets
(32). Hence, we have further investigated the mechanisms
by which TNF- induces inhibition of actin polymerization, and thus
SMCE, by investigating the effect of TNF-
on membrane association of
Ras. In agreement with previous studies in human platelets
(32), we have observed that TG induced translocation of
Ras proteins from the cytosolic to the membrane fraction. In addition,
our results show that treatment for 12 h with TNF-
impaired the
membrane association of Ras stimulated by TG. This process was reversed
by ANP, which had no effect on the membrane association of Ras when
added alone. The results presented here indicate that TNF-
could
exert its effect by inhibiting membrane association of Ras, a process
that is required for the activation of Ras (20), which has
been reported to be important for the reorganization of the actin
cytoskeleton (23, 24, 29, 40) and, subsequently, the
activation of SMCE in HepG2 cells. In addition, we report a protective
role for ANP against these inhibitory effects of TNF-
in HepG2 cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Brown for providing helpful comments while the manuscript was being written and Dr. R. A. Asher for assistance.
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FOOTNOTES |
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* J. A. Rosado and I. Rosenzweig contributed equally to this work.
J. A. Rosado is supported by a Grant of Junta de Extremadura-Consejería de Educación y Juventud and Fondo Social Europeo, Spain.
Address for reprint requests and other correspondence: S. O. Sage, Dept. of Physiology, Downing St., Univ. of Cambridge, Cambridge CB2 3EG, UK (E-mail: sos10{at}cam.ac.uk).
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. Section 1734 solely to indicate this fact.
Received 18 September 2000; accepted in final form 1 December 2000.
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