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Am J Physiol Gastrointest Liver Physiol 293: G552-G559, 2007. First published June 21, 2007; doi:10.1152/ajpgi.00140.2007
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HORMONES AND SIGNALING

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

Lawrence A. Scheving,1,3 Renee Buchanan,1 Michael A. Krause,1 Xiuqi Zhang,1 Mary C. Stevenson,1 and William E. Russell1,2,3,4,5

Departments of 1Pediatrics, Division of Endocrinology and 2Cell and Developmental Biology, 3Digestive Disease Research Center, 4Vanderbilt Diabetes Center, and the 5Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee

Submitted 2 April 2007 ; accepted in final form 18 June 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glucocorticoids paradoxically exert both stimulatory and inhibitory effects on the proliferation of cultured rat hepatocytes. We studied the effects of dexamethasone, a synthetic glucocorticoid, on the proliferation of cultured rat hepatocytes. The timing of growth factor addition modified the action of high-dose dexamethasone (10–6 M) on DNA synthesis. When we added transforming growth factor-{alpha} at the time of plating, 10–6 M dexamethasone weakly stimulated DNA synthesis by 26% relative to cells cultured in dexamethasone-free media. When we delayed growth factor addition until 24–48 h after plating, 10–6 M dexamethasone inhibited DNA synthesis by 50%. Using immunological methods, we analyzed the expression and signaling patterns of the ErbB kinases in dexamethasone-treated cells. High-dose dexamethasone stabilized the expression of epidermal growth factor receptor (EGFr) and ErbB3, and it suppressed the de novo expression of ErbB2 that occurs during the third and fourth day of culture in 10–8 M dexamethasone. High-dose dexamethasone by 72 h suppressed basal and EGF-associated phosphorylation of ERK and Akt. The reduction in ERK1/2 phosphorylation correlated with suppression of a culture-dependent increase in Son-of sevenless 1 (Sos1) and ERK1/2 expression. High-dose dexamethasone in hepatocytes stabilized or upregulated several inhibitory effectors of EGFr/ErbB2 and ERK, including receptor-associated late transducer (RALT) and MKP-1, respectively. Thus 10–6 M dexamethasone exerts a time-dependent and redundant inhibitory effect on EGFr-mediated proliferative signaling in hepatocytes, targeting not only the ErbB proteins but also their various positive and negative effectors.

liver; EGF; TGF-{alpha}; cell culture

DEXAMETHASONE, A SYNTHETIC glucocorticoid, is frequently included in the medium of cultured hepatocytes to improve their survival and function. In most experimental models, dexamethasone inhibits hepatocyte growth in vivo. For example, when injected into the rat, dexamethasone inhibits the striking increase in hepatocyte proliferation that occurs after 70% hepatectomy (28) or in transplanted livers following cold preservation and reperfusion (13). Likewise, dexamethasone has been reported to inhibit the proliferation of rat hepatocytes (34, 35) and liver-derived cell lines (24). In primary hepatocyte culture, however, other investigators have reported that dexamethasone enhances epidermal growth factor (EGF)-stimulated DNA synthesis (26, 30). We hypothesized that these divergent observations result from differential effects of dexamethasone on one or more components of EGF receptor signaling.

Fetal hepatocytes express three of the four ErbB tyrosine kinase receptors (EGFr, ErbB2, and ErbB3), but by 3 wk of age, ErbB2 is largely extinguished (10). When it binds ligands of the EGF/transforming growth factor-{alpha} (TGF-{alpha}) family, EGFr can interact with other ErbB proteins to form activated homo (EGFr/EGFr) or hetero (EGFr/ErbB2 or EGFr/ErbB3) dimers. In contrast, ErbB2 and ErbB3 cannot form active homodimers and must dimerize with each other or with EGFr to form active tyrosine kinase signaling complexes. ErbB2 is structurally unable to bind an extracellular ligand, and ErbB3, which is the receptor for heregulin (Hrg) peptides, has an inactive tyrosine kinase domain. Relative changes in the cell surface expression of individual ErbB proteins can affect cellular signaling outcomes in response to EGF or Hrg peptides (10).

We recently showed that expression of these ErbB proteins changes as hepatocytes adapt to primary culture (10, 31). For example, adult hepatocytes, cultured for 24 h in the presence of insulin and/or EGF, begin to express ErbB2, which is normally expressed in fetal and neonatal but not adult liver (10). EGF- or TGF-{alpha}-stimulated DNA synthesis in adult rat hepatocytes requires the presence of ErbB2 in a heterodimer with EGFr (31). The induction of ErbB2 is the likely explanation for the long lag phase (>40 h from the time of plating) required for hepatocytes to initiate DNA synthesis in response to EGF.

Since the activated glucocorticoid receptor frequently acts as a transcription factor, altering gene expression, we hypothesized that dexamethasone blunts EGF- or TGF-{alpha}-stimulated DNA synthesis by inhibiting the ErbB2 induction. Given the temporal changes of ErbB protein expression in vitro, we further speculated that the overall influence of dexamethasone on the hepatocellular response to EGF-type ligands depends on the time of growth factor addition. Our analysis of prior work suggested that the stimulatory effects of dexamethasone occurred when EGF and dexamethasone were added simultaneously at the beginning of the culture period (30). In contrast, the inhibitory action occurred when isolated hepatocytes were plated in a high concentration of dexamethasone for at least 24 h before EGF exposure (35).

In this paper, we show that the inhibitory effect of dexamethasone on EGF-stimulated DNA synthesis in cultured rat hepatocytes does indeed depend on the time of growth factor addition. Furthermore, our work provides a molecular basis for the inhibitory effect by showing that the concentration of dexamethasone alters not only the expression and interaction of the ErbB proteins themselves, but also a number of positive and negative growth regulatory proteins in ErbB-regulated signaling pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Peptides, reagents, and radiochemicals. Synthetic rat TGF-{alpha} was from Peninsula Laboratories (Belmont, CA). Insulin (Novolin R) was from Novo Nordisk Pharmaceuticals (Princeton, NJ). [3H]thymidine (6 Ci/mmol) was from Perkin Elmer Life Science (Boston, MA). Dexamethasone, pyruvate, bovine serum albumin (fatty acid-free), Percoll, and all buffer reagents were from Sigma (St. Louis, MO). Protein G-Sepharose and ECL reagents were from Pierce (Rockford, IL). Nitrocellulose membranes were from Osmonics (Minnetonka, MN).

Animals. Adult male Sprague-Dawley rats (250–300 g) from Harlan (Indianapolis, IN) were raised under conditions of regulated lighting (lights on 0600–1800). They had ad libitum access to water and Purina rodent chow (Ralston-Purina, St. Louis, MO). The Animal Use Subcommittee of the Vanderbilt Animal Care Committee approved all protocols.

Culture media and supplies. Williams' Medium E, supplemented with 20 mM pyruvate and 50 µg/ml gentamicin, was the medium used for all culture studies. The media typically contained insulin (100 nM), needed to preserve EGF or TGF-{alpha} responsiveness. The concentration of dexamethasone was adjusted for each experiment. Medium and calf serum were from GIBCO, Invitrogen (Grand Island, NY). Type I collagenase was from Wako Pure Chemical Industries (Richmond, VA). Falcon six-well dishes were from Fisher Scientific.

Primary cell cultures. Hepatocytes were isolated from the livers of male rats between 10:00 and 11:30 AM to control for circadian variation by a two-stage, collagenase isolation protocol (9). To reduce nonhepatocyte contamination, cells were sedimented through Percoll (9). We plated cells (3.75 x 105/well) in type 1 collagen-coated six-well 35-mm plates for 60–90 min before adding serum-free medium, growth factors, or dexamethasone. In some experiments, growth factor was added at different times after the change from plating to growth medium.

Immunoprecipitation and Western blotting. Hepatocytes were lysed in TGH buffer (20 mM HEPES, 1% Triton X-100, 10% glycerol 50 mM NaCl). This buffer included protease inhibitors (1 mM PMSF, 1 mM sodium orthovanadate, 10 µg/ml aprotinin, and 1 µg/ml leupeptin) as well as phosphatase inhibitors (10 mM sodium molybdate and 10 mM beta-glycerol phosphate). Lysates were microfuged at 20,800 g for 30 min and then immunoblotted or immunoprecipitated as previously described (4, 10). We used the following affinity-purified antibodies from Santa Cruz Biotechnology (Santa Cruz, CA): sc-03 for EGFR; sc-284 for ErbB2; sc-285 for ErbB3; sc-283 for ErbB4, sc-370 for MKP-1, sc-256 for Son-of sevenless 1 (Sos1), sc258 for Sos2. The anti-phosphotyrosine antibody used was PY99 (Santa Cruz Biotechnology). The anti-phosphotyrosine [Y1112]-ErbB2 antibody was from Orbigen (San Diego, CA). (Brackets represent phosphorylated amino acid numbers.) The anti-phosphotyrosine [Y845]-EGFr, [Y168]-EGFr, [Y1289]-ErbB3, phospho-MEK1/2, phospho-cRaf, the Akt, phospho-Akt and ERK1/2 antibodies were from Cell Signaling Technology (Beverly, MA). The phosphatidylinositol 3-kinase (PI3K) rabbit antiserum (06-195) was from Upstate USA (Charlottesville, VA). The anti-Ras MAb was from Transduction Laboratories (R02120 [GenBank] ).

We normalized immunoprecipitations or immunoblots by using equal amounts of protein, as determined by either the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA) or the Micro BCA protein assay (Pierce Biotechnology). After each transfer, we confirmed equal protein loading and transfer by Ponceau S staining of immunoblots, scanning the image for future reference. Immunoreactive signal was detected by the ECL method (Pierce Biotechnology). We performed densitometry using an Epson scanner with Biosoft Quantiscan Software or the Image J program (1).

DNA synthesis assays. Cells were exposed to the Williams' Medium E for varying periods of time before the addition of TGF-{alpha} (typically at a concentration of 5–7.5 nM); TGF-{alpha} was used in place of EGF because, unlike EGF, which targets EGFr for lysosomal degradation, TGF-{alpha} promotes recycling of internalized EGFr to the hepatocyte surface (31). After stimulation, the cells were refed with medium containing specified growth factors and 1 µCi/ml of [3H-methyl] thymidine. In some experiments, the time of addition of TGF-{alpha} after cell plating varied. After labeling for 24–48 h, cells were harvested, and the incorporation of tracer was determined as detailed previously (9). The results of assays in triplicate are expressed as the specific activity of the DNA (cpm/µg DNA ± SD).

Statistical analysis. Statistical analysis was performed using an unpaired, two-tailed Student's t-test assuming equal variances between compared groups.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Prolonged exposure of hepatocytes to high concentrations of dexamethasone inhibits TGF-{alpha}-stimulated DNA synthesis. To determine the effect of dexamethasone on TGF-{alpha}-stimulated DNA synthesis, we added different concentrations of dexamethasone from 0 to 10–6 M immediately after plating. We then added TGF-{alpha} (1 or 7.5 nM) either at the beginning of the culture period or 48 h later. Figure 1A (left) shows that dexamethasone slightly increased 7.5 nM TGF-{alpha}-stimulated DNA synthesis in a dose-dependent manner when TGF-{alpha} was added immediately after cell plating. For example, in the 7.5 nM TGF-{alpha} treatment groups, dexamethasone at 10–7 M increased DNA synthesis by 26% compared with the dexamethasone-free group (P < 0.05). In contrast, when we delayed growth factor addition until 48 h after plating (right), dexamethasone strongly inhibited TGF-{alpha} stimulated DNA synthesis. Dexamethasone (10–6 M) suppressed 1 nM TGF-{alpha} and 7.5 nM TGF-{alpha}-stimulated DNA synthesis by 78 and 62%, respectively, indicating that the higher concentration of TGF-{alpha} may partly override the action of dexamethasone. The data confirm previous reports that the delayed addition of EGF-like substances elicits a threefold greater peak DNA synthesis in cultured hepatocytes (25) (compare 1 nM TGF-{alpha} at left and right).


Figure 1
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  		Fig. 1. Prolonged dexamethasone exposure inhibits cellular responsiveness to EGFr stimulation. A: effect of dexamethasone on DNA synthesis depends on the time of growth factor addition. TGF-{alpha} was added at either 0 h (left) or 48 h (right). [3H]thymidine incorporation was measured for 48 h beginning at either 48 h (left) or 72 h (right). Dark bars = 7.5 nM TGF-{alpha}; hatched bars = 1.0 nM TGF-{alpha}. B: time course of dexamethasone inhibition. The addition of TGF-{alpha} (7.5 nM) was delayed for up to 48 h in hepatocyte cultures in either 10–6 or 10–8 M dexamethasone. DNA synthesis was assessed between 48 and 96 h of culture. P < 0.05 at 30, 36, and 48 h.

 
To define the kinetics of dexamethasone suppression of EGF-mediated DNA synthesis, we exposed cells to two different concentrations of dexamethasone and then added TGF-{alpha} at different times after plating, evaluating DNA synthesis between 48 and 96 h. Figure 1B shows that dexamethasone did not inhibit TGF-{alpha}-stimulated DNA synthesis during the initial 18 h of culture, indicating that the inhibitory effect of dexamethasone likely required gene transcription. Maximal inhibition occurred when dexamethasone was present for 48 h before TGF-{alpha} addition.

Dexamethasone stabilizes the normal in vivo ErbB expression pattern during cell culture. When placed in culture, adult rat hepatocytes show an altered expression of ErbB proteins. For example, in vivo fetal and neonatal, but not adult, rat hepatocytes normally express ErbB2 (8). However, adult hepatocytes cultured for 48 h reexpress ErbB2, which then contributes to the EGFr signaling complex (31). To determine whether dexamethasone altered the ErbB expression profile, we cultured cells in the presence of 10–8 or 10–6 M dexamethasone for 72 h. We cultured the cells in both insulin (100 nM) and a submitogenic dose of TGF-{alpha} (0.1 nM) to enhance cell viability. Insulin was also added because it is required to preserve maximal TGF-{alpha} responsiveness of hepatocytes after 24 h of culture (30). Lysates were immunoblotted for the four ErbB proteins at 24, 48, and 72 h. Confirming our previous experience, no ErbB4 was detected. Figure 2A shows that the low and high concentrations of dexamethasone had divergent effects on ErbB protein expression. Whereas 10–6 M dexamethasone stabilized the expression of EGFr and ErbB3 between 24 and 72 h, 10–8 M reduced it. The high and low concentrations of dexamethasone also differed in their effect on de novo ErbB2 expression. The overall expression level in the 10–8 M dexamethasone-containing medium consistently exceeded that in the 10–6 M dexamethasone medium. Figure 2B shows the results of quantitative densitometry for ErbB3 and ErbB2 in cells harvested at either 24 or 72 h. Note that there is an inverse expression of ErbB3 (left) and ErbB2 (right) regardless of whether the medium contains dexamethasone at a low or high concentration.


Figure 2
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  		Fig. 2. Dexamethasone (Dex) alters the expression of individual ErbB tyrosine kinase proteins. A: cells were cultured for 24, 48, and 72 h in the presence of either 10–8 or 10–6 M dexamethasone in the presence of a submitogenic dose of TGF-{alpha} (0.1 nM). Cell lysates were immunoblotted for the presence of EGFr, ErbB3, and ErbB2. B: cells were cultured in the presence of either 10–8 or 10–6 M dexamethasone for 24 or 72 h. Lysates were prepared and immunoblotted for ErbB2 or ErbB3 (n = 3 wells). Scanning densitometry was done to show the inverse expression patterns of ErbB2 and ErbB3 at 72 h under conditions of 10–8 or 10–6 M dexamethasone. P < 0.05 for ErbB2 or ErbB3 groups in 10–8 or 10–6 M dexamethasone at 72 h.

 
Dexamethasone modulates EGF-stimulated tyrosine phosphorylation of EGFr and ErbB2. EGF binds EGFr monomers on the cell surface of hepatocytes, leading to the formation of EGFr homodimers or EGFr-ErbB2 or EGFr-ErbB3 heterodimers (10). The data of Fig. 2 show that 10–6 M dexamethasone attenuated the induction of ErbB2. To study the consequence of this on ErbB2 phosphorylation, we exposed cells at either 24 or 72 h of culture to EGF (50 nM) for 1, 3, or 6 min. We then immunoprecipitated ErbB2 and EGFr from soluble lysates. Tyrosine phosphorylation of immunoprecipitated protein was determined by immunoblot with a monoclonal antibody against phosphotyrosine (PY99, IgG2b). We also evaluated the total ErbB2 and EGFr protein immunoprecipitated.

As shown in Fig. 3A (PY row), very little phosphorylated ErbB2 was detected at 24 h, consistent with the very low levels of ErbB2 expression at this time (ErbB2 row). In fact, immunoblot detection of immunoprecipitated ErbB2 with PY-99 or the ErbB2 antibody itself at this time requires prolonged radiographic film exposures (31). In contrast, there was significant tyrosine phosphorylation and expression of ErbB2 at 72 h for cells cultured in 10–8 M dexamethasone. Both phosphorylation and expression were decreased in cells exposed to the 10–6 M concentration. Similar results were obtained for an EGFr immunoprecipitate in a parallel lysate (data not shown).


Figure 3
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  		Fig. 3. Dexamethasone inhibits EGF stimulated tyrosine phosphorylation of ErbB2. A: cells were cultured in the presence of either 10–8 or 10–6 M dexamethasone for 24 or 72 h. The cells were then exposed to EGF (100 nM) for 0, 1.5, 3, or 6 min. ErbB2 was immunoprecipitated from Triton X-100 solubilized cell lysates and then probed with a monoclonal antibody against phosphotyrosine [PY99 (PY); top]. Equal loading was shown by stripping the blot and probing it with an ErbB2 polyclonal antibody (bottom). B: cells were cultured in the presence of either 10–8 or 10–6 M dexamethasone for 24 or 72 h. The cells were then treated with EGF (10 nM) for 0, 1, or 3 min and then analyzed for phosphorylated EGFr (top) or ErbB2 (bottom) by immunoblot using phospho-EGFr or ErbB2 antibodies.

 
To confirm these effects, we used antibodies that specifically recognize discrete tyrosine phosphorylation sites (pY) within the cytoplasmic domains of EGFr or ErbB2. We used antibodies that recognize pY845 and pY1068 for EGFr and pY1112 for ErB2. Parallel blots were performed to find out whether the degree of tyrosine phosphorylation correlated to the total ErbB expression level. Figure 3B shows the results. At 24 h, for both EGFr and ErbB2 the amount of acute tyrosine phosphorylation in response to exogenous EGF (10 nM) was slightly greater in the cells treated with 10–6 M compared with 10–8 M (data were reproducible in multiple experiments). In contrast, for both of these interactive ErbBs, the degree of tyrosine phosphorylation at 72 h in the cells cultured in 10–8 M dexamethasone greatly exceeded those cultured in 10–6 M. After normalization for overall ErbB2 levels, the specific phosphotyrosine signal in ErbB2 immunoprecipitates from the 10–8 M dexamethasone exceeded that of ErbB2 immunoprecipitates from the cells treated in 10–6 by twofold (data not shown). These data suggest that high-dose dexamethasone exerts an inhibitory influence on EGF-stimulated hepatocyte DNA synthesis by preventing the expression and phosphorylation of ErbB2.

Dexamethasone alters ERK1/2 signaling. After showing that the level of ErbB phosphorylation varied in the dexamethasone-treated cells, we evaluated the expression level and phosphorylation state of components of the ERK1/2 pathway, which is one of the two signaling pathways required for EGF to exert its full mitogenic effect in hepatocytes (33). As shown in Fig. 4, dexamethasone had little or no effect on the expression level of Ras at 24 or 72 h of cell culture. Dexamethasone suppressed the two Son-of-sevenless (Sos) guanine nucleotide exchange factors that interact with Grb2 upon growth factor binding. These two proteins showed an interesting reciprocal pattern of expression with time in culture. Sos2 predominated at 24 h whereas Sos1 was strongly induced by 72 h of cell culture, when Sos2 disappears.


Figure 4
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  		Fig. 4. Dexamethasone alters ERK1/2 signaling. A: cells were treated with different concentrations of dexamethasone for 24 or 72 h and evaluated for the total protein expression of Grb2, Son-of sevenless 1 (Sos1), Sos2, and RAS. Note that Sos1 increases with culture time, but the higher concentrations of dexamethasone suppress this increase. B: cells were treated as described in A and then evaluated by immunoblot for the phosphorylated forms of Raf, MEK1/2, ERK1/2, or total ERK1/2. Note that the total amount of ERK1 increases with culture time, although dexamethasone suppresses this increase. There is increased baseline phosphorylation of Raf, MEK1/2, and ERK1/2 in the cells in the dexamethasone-free or 10–8 M dexamethasone for 72 h. C: Cells were cultured (n = 3 wells) in the presence of increasing concentrations of TGF-{alpha} (0, 0.1, 1, or 5 nM) in 10–8 or 10–6 M dexamethasone. Immunoblots were analyzed by scanning densitometry for ErbB2 (left) and ERK1/2 (right) at 72 h. Note that 10–6 M dexamethasone diminishes ErbB2 and ERK1/2 expression.

 
Moreover, when we examined the phosphorylation state of Raf (Ser338), MEK1/2 (Ser217/221), and ERK1/2 (Thr202/Tyr204) by use of phospho-specific antibodies, we observed that dexamethasone-free or low-dexamethasone concentrations were associated at 72 h with higher baseline levels of the phosphorylated species. Moreover, the increase in the active ERK1/2 species partially correlated with an increase in total ERK1/2.

To explore the ability of dexamethasone to alter the responsiveness of hepatocytes to EGFr signaling, we cultured cells in the presence of increasing concentrations of TGF-{alpha} instead of a single submitogenic dose (0.1 nM) as above. Figure 4C shows that after 72 h, cells cultured in the presence of 10–8 M dexamethasone had higher levels of ErbB2 (left) and ppERK1/2 (right) than cells cultured in the 10–6 M concentration. The overall levels of both ErbB2 and ppERK1/2 increased with increasing concentrations of TGF-{alpha}.

Dexamethasone inhibits EGF stimulation of ERK1/2 and PI-3 kinase. Phospho-Akt is an indirect measure of the PI3K pathway, a second pathway required for full mitogenic action of EGF (8, 33). In the prior experiment, we evaluated whether dexamethasone altered phospho-Akt and were unable to find a consistent effect (data not shown). Since the cells in the prior experiment had not been exposed to fresh TGF-{alpha} for over 24 h (the TGF-{alpha} was added at time 0 for the 24-h group and 48 h for the 72-h group), we analyzed the acute effect of ligand on Akt phosphorylation in cells cultured in different concentrations of dexamethasone. We examined the acute response of ERK1/2 to EGF in this experiment as well.

In Fig. 5, we stimulated cells with EGF and under conditions of low or high dexamethasone for 24 or 72 h. We then immunoblotted lysates for the total and phosphorylated forms of ERK1/2 or the phosphorylated Akt (Ser473). As shown in Fig. 5, irrespective of the dexamethasone concentration, cells cultured for 72 h showed a remarkable increase in the relative amount of total Akt and ERK1/2 compared with 24 h.


Figure 5
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  		Fig. 5. Dexamethasone regulates acute EGF-dependent phosphorylation of Akt and ERK1/2. We treated cells with either 10–8 or 10–6 M dexamethasone for 24 or 72 h and then exposed them to EGF (50 nM) for 0, 1.5, 3, or 6 min. This figure shows a representative experiment. Note that 10–6 M dexamethasone increases the total amount of Akt and ERK at 24 h. Both Akt and ERK increase at 72 h, but the increase in the total amount of ERK1, but not ERK2 or Akt, is suppressed by the highest concentration of dexamethasone. All of the phosphorylated forms of ERK1/2 and Akt are diminished in cells cultured in the highest concentrations of dexamethasone at 72 h, which indicates that other mechanisms besides protein density account for the inhibitory effect of dexamethasone in this model.

 
When cultured in 10–8 M or 10–6 M dexamethasone for 24 h, cells exposed to the highest concentration showed a greater activation of ERK1/2 and Akt that partly correlated with increased expression levels of these molecules at this time. In contrast, by 72 h, cells cultured in the high concentration showed decreased levels of phosphorylated Akt and ERK1/2. Again, the reduced phosphorylation partly correlated with a reduction in the total protein, particularly for ERK1/2.

Dexamethasone induces molecules that inhibit ErbB signaling. Dexamethasone inhibits proliferation of a number of other cell types using negative effectors, including receptor-associated late transducer (RALT) (2), MKP-1 (14), and the p85 regulatory subunit of PI3K (17). We evaluated their expression in hepatocytes as a function of the concentration of dexamethasone (Fig. 6). We found that 10–6 M dexamethasone stabilized the expression of RALT, which normally disappears by 48 h in the absence of dexamethasone. We observed that increasing concentrations of dexamethasone suppressed the expression of the p85 regulatory subunit of PI3K at 24 h of culture. However, this effect was reversed by 72 h. Finally, we found that dexamethasone modulated MKP-1. In contrast to RALT and p85, hepatocytes express little or no MKP-1 unless they are cultured in dexamethasone, which increases MKP-1 in a dose-dependent manner.


Figure 6
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  		Fig. 6. Dexamethasone modulates inhibitors of MAPK and phosphatidylinositol 3-kinase (PI3K)-signaling pathways. We treated cells for 24, 48, or 72 h with different concentrations of dexamethasone and evaluated the total protein expression RALT (left), MKP-1 (middle), and the 85-kDa regulatory subunit of PI3K (right). Note higher concentrations of dexamethasone stabilize the expression of RALT while inducing that of MKP-1. Dexamethasone is required for p85 expression at 72 h but suppresses its expression at 24 h in a dose-dependent manner.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Dexamethasone has complex effects on cell proliferation in different tissues and cell types (36). It may augment the effect of a specific growth factor on DNA synthesis in some cell types but inhibit it in others. For example, it enhances the stimulatory effect of EGF in myoblasts (20) and human diploid fibroblasts but inhibits the response in 3T3 cells (5). In a given cell type, the actions of dexamethasone can vary between growth factors. In 3T3 cells, dexamethasone inhibits the growth promoting action of EGF but potentiates that of fibroblast growth factor (18, 19). This suggests that the action of dexamethasone does not target a single cytoplasmic or nuclear mechanism distal to the interaction between growth factors and their cell surface receptors.

Many glucocorticoid effects are both time and transcription dependent. However, some actions that depend on glucocorticoid binding to its receptor do not require subsequent binding of the receptor to glucocorticoid response elements within DNA (6, 7, 22). For example, in the A549 human lung adenocarcinoma cell line, dexamethasone rapidly inhibits recruitment of signaling factors to EGF receptors, increasing the tyrosine phosphorylation of lipocortin in a transcription-independent manner. The phosphorylated lipocortin binds to GrbB2 docking sites on EGFr, thereby inhibiting the transmission of signals necessary for EGF-stimulated arachidonic acid release and cell growth (12).

The liver of the intact rat is exquisitely sensitive to the inhibitory actions of dexamethasone and other glucocorticoids, such as hydrocortisone, on cell proliferation. These hormones strongly inhibit DNA synthesis in the developing or regenerating liver and in all liver-derived cell lines (24). For example, hydrocortisone completely blocks DNA synthesis within a 24-h period when injected into weaning rats. It also partially inhibits DNA synthesis in the regenerating liver of young rats after partial hepatectomy, blunting the regenerative response by ~50%. Recently, it has been shown that dexamethasone inhibits the early regenerative response of the rat liver seen after cold preservation and transplantation (13). Furthermore, Nagy et al. (28) demonstrated that daily injections of dexamethasone (2 mg/kg) immediately before and after 70% hepatectomy in the rat inhibited not only hepatocyte proliferation but also oval cell proliferation and differentiation. Interestingly, in this model, the liver restored its mass by the preferential hypertrophy of periportal hepatocytes. The action of dexamethasone was reversible. Forty hours after dexamethasone withdrawal, the enlarged periportal cells entered the S phase in a synchronized fashion.

Despite these growth inhibitory effects in the liver from the intact rat, dexamethasone or other glucocorticoids are usually included in the growth medium of cultured hepatocytes because they increase cell attachment and survival. They inhibit spontaneous apoptosis through the induction of Bcl-2 and Bcl-xL (4), and they also inhibit the TNF and Fas death receptor-mediated apoptosis through the upregulation of Flice inhibitory protein (cFlip) (29). Some investigators have reported that dexamethasone increases EGF-stimulated DNA synthesis (26, 30), but others have reported that dexamethasone inhibits it (34, 35). These divergent effects are unrelated to differences in dose since they both varied in a dose-dependent manner. No clear concept to explain these discrepant findings has arisen until now.

In this paper, we show that the timing of the growth factor addition relative to the initial dexamethasone exposure is the critical determinant in defining whether dexamethasone has stimulatory or inhibitory effects on DNA synthesis (Fig. 1A). This conclusion is supported by the analysis of conflicting papers. Sand et al. (30) showed that, in hepatocytes treated with insulin and EGF from the time of plating, dexamethasone increased the rate of DNA synthesis by 20–30%. In contrast, Vintermyr and Doskeland (34) reported that when cells were exposed to EGF after 20 h of culture DNA synthesis was inhibited by dexamethasone. The latter authors speculated that the use of collagen-coated plates or the inclusion of pyruvate in their media may have been responsible for the discrepancies. We now show that neither collagen nor pyruvate can account for them since they are also components of our culture system. Instead, the inhibitory effect of dexamethasone is not immediate as described for some cell types (12) but requires cell exposure to dexamethasone for as long as 18 h before growth factor addition (Fig. 1B).

Very little is known about the mechanisms used by dexamethasone to stimulate or inhibit DNA synthesis in the intact liver and in primary hepatocytes. Several possible explanations have been offered for the inhibitory effect on hepatocytes, including the synthesis and release of a secreted growth inhibitory factor or a stabilizing effect on gap junctions and cell contacts, which decrease during states of high cell proliferation, such as liver regeneration (34). Indeed, others reported that the inhibitory effect of dexamethasone was greater in areas of high cell density, suggesting that cell contact contributed to the effect (34).

We have recently shown that the ErbB expression profile in hepatocytes changes during adaptation to cell culture (31), coinciding with an increased responsiveness to EGF (25). The proliferative effects of EGF in cultured hepatocytes require the induction of ErbB2 shortly before DNA synthesis rises 48 h after plating. Since the timing of growth factor addition modulated the inhibitory glucocorticoid effect on DNA synthesis (Fig. 1), we speculated that dexamethasone altered either the expression of the ErbB proteins or downstream signaling pathways. Indeed, Baker et al. (5) reported a precedent for a dexamethasone-induced growth-altering change in the cell surface expression of growth factor receptors. In human diploid fibroblasts, dexamethasone enhanced EGF-stimulated DNA synthesis while increasing 125I-EGF binding to surface membranes by 1.5-fold at 8 h after addition and by twofold at 32 h.

We now report that dexamethasone has far-reaching effects on ErbB expression and activation (Figs. 2 and 3). Moreover, these changes in ErbB expression resulted in a decrease in EGF-stimulated tyrosine phosphorylation of EGFr and ErbB2 (Fig. 3). Dexamethasone also altered signaling downstream of the ErbB proteins, including the ERK and PI3K pathways (Figs. 4 and 5). Both pathways are required for EGF to stimulate DNA synthesis in cultured hepatocytes, as indicated by studies using pharmacological or genetic inhibitors (8, 33). Dexamethasone at a high dose suppressed both pathways in cells treated for 72 h of culture.

Other groups have shown that dexamethasone suppresses these pathways in other cell types by upregulating negative effectors that either directly bind to the ErbB proteins or indirectly inhibit ErbB action by inhibiting ERK or PI3K. Dexamethasone can induce unique adaptor-like molecules that bind to discrete regions of the ErbB cytoplasmic domains and inhibit signaling. Such proteins include RALT (also known as gene 33 and MiG-6) and Dok-1 (21). RALT suppresses the tyrosine kinase activity of both ErbB-2 and EGFR (2, 15, 16, 38). Dexamethasone also inhibits signaling components downstream from the ligand-ErbB interaction. Inhibition of ERK primarily occurs through the induction of the dual-specificity phosphatase MKP-1, which dephosphorylates phosphoserine and phosphothreonine residues within ERK, attenuating the transmission of growth factor signals (14, 23). Dexamethasone also exerts some of its antiproliferative and proapoptotic effects in certain cell types by upregulating the p85 regulatory subunit and thereby inhibiting PI3K (11, 17, 32). In hepatocytes, we found that dexamethasone influenced the expression of p85, RALT, and MKP-1 in a manner that generally correlated with its inhibitory effect on DNA synthesis (Fig. 6).

Hepatocytes in primary culture are a popular model to study and define the regulation of hepatocyte growth, function, and signaling. However, primary hepatocytes are a dynamic system that adapt to the conditions of culture. In doing so, they undergo striking changes in the expression patterns of cell surface receptors, such as the ErbB proteins (Figs. 2 and 3); downstream growth promoter elements, such as ERK and Akt (Figs. 4 and 5); and growth inhibitors, such as RALT and MKP-1 (Fig. 6). These changes may lead to conflicting results, particularly when cells cannot be used for a period of time so as to induce or knock down gene expression experimentally. Our present work shows how consideration of this factor reconciles contradictory reports that dexamethasone stimulates or inhibits EGF-stimulated hepatocyte proliferation. In this paper, we have shown that the ability of dexamethasone to stimulate or inhibit proliferation is highly dependent on the time that EGF ligands are introduced into culture. Dexamethasone augments EGF-stimulated DNA synthesis when dexamethasone and EGF are added together shortly after cell isolation and plating. In contrast, prolonged exposure (>24 h) to high concentrations of dexamethasone before ligand activation of EGFr inhibits hepatocyte DNA synthesis. Dexamethasone in this instance causes extensive changes in cell signaling, curbing numerous culture-dependent changes in the ErbB proteins and their effectors that collectively optimize the hepatocellular proliferative response to EGF.

The ability of dexamethasone to regulate in a coordinate fashion the many positive and negative signaling elements involved in ErbB signaling may be a consequence of its effects on differentiation. For example, dexamethasone alone is sufficient to transform pancreatic cells into hepatocyte-like cells that can support replication of hepatitis B virus (37). Dexamethasone also has dramatic effects on hepatocyte differentiation in organoid cultures, suppressing growth and inducing hepatocyte maturation (27). Indeed, dexamethasone activates an enhancer element 6 kb upstream of the HNF4{alpha}-1 promoter that positively regulates the expression of this transcription factor, which is weakly expressed in the embryonic liver and strongly expressed in the adult liver (3). The relative contributions of individual ErbB signaling elements in the actions of dexamethasone require further study. Indeed, some of the effects of dexamethasone on DNA synthesis may proceed through other complex signaling pathways not monitored in the present work. Future work involving direct modulation of various signaling proteins may allow us to define their relative importance in mediating the effects of dexamethasone on DNA synthesis.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge the generous support of student research stipend to R. Buchanan from the Vanderbilt Diabetes Research and Training Center (DK-20593) and Vanderbilt Short-Term Research Training Program for Medical Students (T35DK007383). This work was supported by DK-53804 (to W. E. Russell).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Scheving, Division of Pediatric Endocrinology, 1055 Medical Research Bldg. 4, Vanderbilt Univ. Medical Center, Nashville, TN 37232-0710 (e-mail: lawrence.a.scheving{at}vanderbilt.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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