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Tumor necrosis factor-alpha inhibits store-mediated Ca2+ entry in the human hepatocellular carcinoma cell line HepG2

Juan A. Rosado*, Ivana Rosenzweig*, Susanne Harding, and Stewart O. Sage

Department of Physiology, University of Cambridge, Cambridge CB2 3EG, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Tumor necrosis factor-alpha (TNF-alpha ) 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-alpha 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-alpha slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-alpha 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-alpha abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-alpha was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-alpha 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-alpha .

calcium influx; actin cytoskeleton; Ras proteins; atrial natriuretic peptide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SEVERAL STUDIES HAVE REPORTED that the pleiotropic cytokine tumor necrosis factor-alpha (TNF-alpha ) 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-alpha recognizes two cell surface receptors that could mediate different cellular effects sheds light on the understanding of the biological activity of TNF-alpha (8). In liver cells, TNF-alpha 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-alpha is also known to induce apoptosis in hepatocytes (15). Several studies have suggested a close relationship between TNF-alpha 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-alpha decreased thapsigargin (TG)-induced Ca2+ entry in thyroid FRTL-5 cells, although the mechanism of action of TNF-alpha 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-alpha -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-alpha -evoked inhibition.


    MATERIALS AND METHODS
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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-alpha , 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 alpha -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 alpha -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.


    RESULTS
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RESULTS
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Regulation of ANP immunoreactivity by TNF-alpha . HepG2 cells were treated with TNF-alpha (100 ng/ml) or the vehicle for 12 h and then incubated with anti-alpha -ANP antibody to identify alpha -ANP and also with Hoechst 33342 to identify the nucleus of the cells. Treatment of HepG2 cells for 12 h with TNF-alpha (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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Fig. 1.   Regulation of atrial natriuretic peptide (ANP) immunoreactivity in HepG2 cells by tumor necrosis factor-alpha (TNF-alpha ). HepG2 cells were incubated for 12 h with either 100 ng/ml TNF-alpha (ii) or vehicle (i). Cells were then immunostained with both the rabbit polyclonal ANP antibody (A) and the DNA-binding dye Hoechst 33342 (B), as described in MATERIALS AND METHODS (n = 5). Scale bar, 50 µm.

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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Fig. 2.   Thapsigargin (TG) stimulates divalent cation entry. A: fura 2-loaded HepG2 cells were resuspended in Ca2+-free HEPES-buffered saline. At the time of the experiments, 100 µM EGTA was added. Cells were then treated with 3 µM TG or vehicle (control), and 3 min later CaCl2 (final concentration 5 mM) was added to the medium to initiate Ca2+ entry. B: fura 2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. HepG2 cells were treated with 3 µM TG or vehicle (control) 3 min before the addition of MnCl2 (final concentration 5 mM). Traces are representative of 4 experiments. [Ca2+]i, intracellular Ca2+ concentration.

Mn2+ was used to evaluate the effect of TG on bivalent cation entry in HepG2 cells. This cation can be used as a surrogate for Ca2+ entry, given its quenching effect on fura 2 (36). Fura 2 was excited at the isoemissive wavelength, 360 nm, to permit the monitoring of quenching of fluorescence by Mn2+. As shown in Fig. 2B, the addition of Mn2+ (5 mM) to HepG2 cells with TG-depleted intracellular Ca2+ stores resulted in a sustained quenching of fluorescence compared with that in undepleted cells.

Effect of TNF-alpha , ANP, or a combination of both on SMCE in HepG2 cells. Treatment of cells for 10 min with 100 ng/ml TNF-alpha 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-alpha (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-alpha when the two agents were added together. ANP reversed the inhibitory effect of TNF-alpha on SMCE to 63 ± 6% of control (Fig. 4; P < 0.05; n = 8).


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Fig. 3.   Effects of short-term exposure to TNF-alpha , ANP, or both on store-mediated Ca2+ entry (SMCE) in HepG2 cells. A: fura 2-loaded HepG2 cells were incubated for 10 min the presence of 100 ng/ml TNF-alpha , 100 ng/ml TNF-alpha  + 1 µM ANP, or vehicles (control). At the time of the experiments, 100 µM EGTA was added. Cells were then stimulated with TG (3 µM), and 3 min later CaCl2 (final concentration 5 mM) was added to the medium to initiate Ca2+ entry. B: histograms indicate the percentage of Ca2+ entry after the different treatments relative to their controls (vehicle was added). Ca2+ entry was determined as described in MATERIALS AND METHODS. Values are means ± SE of 8 independent experiments and are expressed as a percentage of Ca2+ entry in controls (TG-stimulated nontreated cells). *P < 0.05, compared with control.



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Fig. 4.   Effects of long-term exposure to TNF-alpha or TNF-alpha  + ANP on SMCE in HepG2 cells. A: HepG2 cells were incubated for 12 h in the presence of 100 ng/ml TNF-alpha , 100 ng/ml TNF-alpha  + 1 µM ANP, or vehicles (control) and loaded with fura 2 as described in MATERIALS AND METHODS. At the time of the experiment, 100 µM EGTA was added. Cells were then stimulated with TG (3 µM), and 3 min later CaCl2 (final concentration 5 mM) was added to the medium to initiate Ca2+ entry. Traces are representative of 8 independent experiments. B: histograms indicate the percentage of Ca2+ entry after the different treatments relative to their controls. Ca2+ entry was determined as described in MATERIALS AND METHODS. Values are means ± SE. *P < 0.05, **P < 0.01, compared with control.

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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Table 1.   Effects of cytochalasin D or latrunculin A on the F-actin content of unstimulated and TG-stimulated HepG2 cells

FITC-phalloidin labeling revealed an organized actin filament network when the cells were observed by confocal microscopy (Fig. 5A). Consistent with the results reported above, treatment of HepG2 cells for 1 h with 10 µM Cyt D induced almost complete disappearance of the stress fibers (Fig. 5B). In addition, the actin filament content of the cells appeared to be substantially reduced compared with that of control cells. As shown in Fig. 5C, treatment of HepG2 cells with 3 µM Lat A for 1 h resulted in similar cytoskeletal changes.


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Fig. 5.   Cytochalasin D (Cyt D) and latrunculin A (Lat A) induced disassembly of the actin cytoskeleton. HepG2 cells were left untreated (A) or incubated for 1 h with 10 µM Cyt D (B) or 3 µM Lat A (C). F-actin was stained with FITC-labeled phalloidin, and cells were examined by confocal fluorescence microscopy. Representative fields are shown. Bars, 10 µm.

Treatment of HepG2 cells at 37°C for 1 h with 10 µM Cyt D or 3 µM Lat A abolished TG-evoked Ca2+ entry (Fig. 6; P < 0.001; n = 5). Cyt D- or Lat A-treated cells showed an identical release of Ca2+ from the internal stores upon stimulation with TG compared with untreated cells, indicating that accumulation of Ca2+ in the internal stores was unaffected by inhibition of actin polymerization (Fig. 6).


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Fig. 6.   Effect of Cyt D and Lat A on TG-evoked Ca2+ entry in HepG2 cells. Fura 2-loaded HepG2 cells were incubated for 1 h in the presence of 10 µM Cyt D (A), 3 µM Lat A (B), or vehicles (control). At the time of the experiment, 100 µM EGTA was added. Cells were then stimulated with TG (3 µM), and 3 min later CaCl2 (final concentration 5 mM) was added to the medium to initiate Ca2+ entry. Traces are representative of 5 independent experiments.

Effect of TNF-alpha , ANP, or a combination of both on TG-induced actin polymerization in HepG2 cells. To investigate whether the inhibitory effect of TNF-alpha on SMCE is mediated by interference with actin polymerization, we pretreated HepG2 cells with 100 ng/ml TNF-alpha for 12 h and determined actin filament content using FITC-phalloidin. As shown in Fig. 7A, long-term exposure to TNF-alpha abolished TG-stimulated actin filament formation in HepG2 cells (P < 0.001; n = 5). TNF-alpha 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-alpha on TG-induced actin polymerization; the effect was very similar to that which ANP had on SMCE when added together with TNF-alpha (Fig. 7A; P < 0.05; n = 5).


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Fig. 7.   Effects of TNF-alpha , ANP, or both on the actin filament content of TG-treated HepG2 cells. HepG2 cells were treated for 12 h (A and B) or for 10 min (C) with 100 ng/ml TNF-alpha , 1 µM ANP, or both and then loaded with dimethyl 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (B) or left unloaded (A and C). Cells were then stimulated with TG (3 µM) in a Ca2+-free medium (100 µM EGTA was added), and samples were removed 5 s before and 3 min after TG was added. Actin filament content was determined as described in MATERIALS AND METHODS. Values means ± SE of 6 separate determinations and represent TG-evoked actin filament formation expressed as a percentage of control (TG-stimulated nontreated cells). ***P < 0.001, compared with F-actin content in TG-stimulated untreated cells.

Similar experiments were performed on cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator, to eliminate Ca2+-dependent but not store depletion-dependent responses (37). Dimethyl BAPTA loading reduced TG-induced actin polymerization by 28 ± 2% (not shown). As shown in Fig. 7B, the effect of 12 h of exposure of dimethyl BAPTA-loaded cells to TNF-alpha , ANP, or both was similar to the effect on non-BAPTA-loaded cells. Long-term exposure to TNF-alpha almost completely inhibited TG-stimulated actin filament formation (P < 0.001; n = 5) and slightly reduced the actin filament content of unstimulated cells by 11 ± 7% (n = 5). Exposure to ANP for 12 h had no significant effect on either TG-induced actin polymerization (Fig. 7B; P = 0.57; n = 5) or the actin filament content in unstimulated cells (data not shown) but significantly reversed the inhibitory effect of TNF-alpha (Fig. 7B, P < 0.05; n = 5).

Consistent with the effect observed on SMCE, treatment of HepG2 cells for 10 min with 100 ng/ml TNF-alpha increased TG-induced polymerization, although this effect was not significant (Fig. 7C; P = 0.17; n = 4), without having any effect on resting actin filament content (not shown). Treatment with 1 µM ANP or both TNF-alpha and ANP had no effect on either the actin filament content of unstimulated (data not shown) or TG-stimulated cells (Fig. 7C; n = 4).

Effect of TNF-alpha , 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-alpha 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-alpha , 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-alpha on Ras association with membranes (Fig. 8; P < 0.01; n = 3). Treatment for up to 12 h with TNF-alpha , ANP, or both had no effect on the cellular distribution of Ras in unstimulated HepG2 cells (data not shown).


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Fig. 8.   Effect of TNF-alpha on the membrane association of Ras proteins in HepG2 cells. HepG2 cells were treated for 12 h with no addition or with 100 ng/ml TNF-alpha , 1 µM ANP, or both. HepG2 cells loaded with dimethyl BAPTA were then stimulated with TG (3 µM) in a Ca2+-free medium. Samples were taken 5 s before and 3 min after TG was added. Cytosolic fractions (C) and membrane fractions (M) were isolated as described in MATERIALS AND METHODS. Lysates of the subcellular fractions (20 µg/well) were analyzed by Western blotting with a pan-Ras antibody. Arrow represents the position of the putative Ras proteins. Histogram results are representative of 3 separate experiments. ** P < 0.01, *** P < 0.001 (Student's t-test for paired samples).


    DISCUSSION
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TNF-alpha 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-alpha resulted in inhibition of SMCE. TNF-alpha selectively inhibited TG-induced SMCE without having any effect on the release of Ca2+ from the intracellular stores, which indicates that TNF-alpha did not affect the ability of HepG2 cells to store Ca2+ in intracellular compartments. The inhibition of Ca2+ entry by TNF-alpha 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-alpha .

A number of studies have shown that TNF-alpha 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-alpha 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-alpha synthesis (4, 19). Interestingly, we found that 12 h of incubation of cells with TNF-alpha 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-alpha mRNA during reperfusion of the rat liver. Also, we have recently reported (33) that ANP was able to partially protect hepatocytes against TNF-alpha -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-alpha and ANP, the inhibition of Ca2+ entry observed when cells were treated with TNF-alpha alone was significantly reduced, indicating that ANP has a protective effect against TNF-alpha -induced inhibition of SMCE. In agreement with our findings, the effect of ANP in modulating TNF-alpha -induced responses has been recently shown in the human renal proximal tubule, where ANP inhibited TNF-alpha -stimulated nitric oxide production (6).

It remains to be fully elucidated how TNF-alpha 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-alpha and ANP on actin filament formation in these cells. Our results show that long-term exposure of cells to TNF-alpha 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-alpha 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-alpha -induced inhibition of actin polymerization. As shown for the Ca2+ entry mechanism, 10 min of preincubation with TNF-alpha 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-alpha -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-alpha 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-alpha induces inhibition of actin polymerization, and thus SMCE, by investigating the effect of TNF-alpha 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-alpha 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-alpha 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-alpha in HepG2 cells.


    ACKNOWLEDGEMENTS

We thank Dr. John Brown for providing helpful comments while the manuscript was being written and Dr. R. A. Asher for assistance.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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