Early effect of growth factors (EGF+insulin) upon ATP turnover in 3T3 cells

Various early biochemical events have been observed after the addition of growth factors to quiescent cultures of 3T3 cells; however, the cascade of events which take place in the cells after growth-factor addition is not yet entirely known. Our results show that the addition of a mixture of two growth factors, i.e., Epidermal Growth Factor (EGF) and insulin, to quiescent cultures of 3T3 cells rapidly stimulated phosphate uptake and ATP turnover. Our present and previous results suggest that the increase in phosphate uptake is the consequence of the stimulation of ATP synthesis. This stimulation was not simply a consequence of an increase in oxidative phosphorylation or in glucose transport and metabolism. The change in ATP turnover was an early event observed as soon as 5 min after growth-factor addition; furthermore, it was not dependent on protein synthesis. This change may therefore be the result of post-synthetic modification of enzymes by phosphorylation. We do not know what cellular process is responsible for the increase in ATP turnover. Since growth-factor addition rapidly enhanced ATP degradation in quiescent 3T3 cell cultures, we assumed that this increase is the result of an increase in ATP degradation. We know that it was not due to a stimulation of an oligomycin-sensitive ATPase. We verified that it was not the consequence of early biochemical events like an increase in Na+/K+ ATPase or a stimulation of RNA or protein synthesis. However, it is of interest to note that the stimulation of ATP turnover due to the growth-factor addition was inhibited by quercetin.     

Early effect of growth factors (EGF + insulin) upon ATP turnover in 3T3 cells. Inhibition by cytochalasin B and D

We have demonstrated previously a rapid increase in ATP turnover soon after adding epidermal growth factor (EGF) and insulin to quiescent cultures of Swiss 3T3 cells. In the present work, we tried to determine whether this increase could be correlated with the early stimulation by growth factors of cell movements. We showed that cytochalasin B (CB), in complete or glucose-free medium, inhibited this early increase caused by growth factors, in phosphate incorporation in small organic acid-soluble compounds (Po). Cytochalasin D (CD) specifically inhibited the stimulation caused by growth factors of Po labelling and ATP turnover, but lacked all inhibitory effect on unstimulated cells. The inhibitory effect of CD was transient. We hypothesize that addition of EGF and insulin to quiescent 3T3 cell cultures induces a rapid and transient change in cell movements, which could be responsible for about half of the early increase in ATP degradation and turnover.     

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.     

Anisomycin and cycloheximide, like growth factors, stimulate rapidly ATP turnover in 3T3 cells

Addition of EGF and insulin to quiescent cultures of 3T3 cells induce a rapid stimulation of ATP turnover. We show that, like growth factors, anisomycin and cycloheximide, two inhibitors of protein synthesis, induce in 3T3 cells, a rapid increase in ATP turnover. However, this effect was not the result of the inhibition of protein synthesis since puromycin, in the same experimental conditions, did not stimulate ATP turnover.     

Early stimulation of ATP turnover by EGF + insulin. Relation to external pH and Na+/H+ exchange system

We previously shown a rapid increase in ATP turnover after addition of epidermal growth factor and insulin to quiescent 3T3 cell cultures. Here, the relationship between this increase in ATP turnover and the activation by growth factors of Na+/H+ and Na+/K+ exchange systems was studied. Our results show that alkalinization of the medium enhances ATP turnover but they do not support the assumption that stimulation by growth factors of the Na+/H+ exchange induces an increase in ATP turnover since this increase was not inhibited by amiloride. Conversely, when ATP synthesis was abolished, the increase, in intracellular pH, by growth factors, was significantly decreased.     

Activation of MAP kinase and enhanced phosphorylation of the 350-kDa protein by mitogenic stimuli in quiescent Balb/c 3T3 cells.

In quiescent Balb/c 3T3 cells, competence factors such as platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate (TPA) activated MAP kinase, whereas progression factors such as insulin did not. Insulin was, however, capable of activating MAP kinase in cells pretreated with TPA. Moreover, TPA plus insulin activated MAP kinase more strongly and for a longer time period than did TPA alone. Treatment of Balb/c 3T3 cells with competence factors stimulated phosphorylation of the 350-kDa protein which was immunoprecipitated with antibodies against brain high-molecular-weight microtubule-associated protein MAP1, whereas insulin treatment did not stimulate the phosphorylation. Insulin could induce, however, further increase in the phosphorylation of the 350-kDa protein, when added simultaneously with TPA or added to the TPA-treated cells. The enhanced phosphorylation of the 350-kDa protein thus correlated with the MAP kinase activation. As insulin acts synergistically with TPA to induce initiation of DNA synthesis in the quiescent Balb/c 3T3 cells, it seems that activation of MAP kinase and enhanced phosphorylation of the 350-kDa protein are accompanied by the initiation of DNA synthesis.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.     

Stimulation of DNA synthesis in quiescent rabbit chondrocytes in culture by limited exposure to somatomedin-like growth factors

Cartilage-derived factor (CDF), extracted from fetal bovine cartilage, and multiplication-stimulating activity (MSA) stimulated DNA synthesis in quiescent rabbit costal chondrocytes in culture under serum-free conditions. As described previously, when added in the presence of fibroblast growth factor (FGF) or epidermal growth factor (EGF) a somatomedin-like growth factor, CDF or MSA, synergistically stimulated DNA synthesis in the cultured chondrocytes. The present study showed that exposure of the cells to MSA or CDF for only the initial 5 h was sufficient for transmission of their full stimulatory effect. Furthermore, the limited exposure did not alter the time course of stimulation of DNA synthesis: [3H]thymidine incorporation into DNA began to increase after 16 h and reached a maximum after 24 h. In contrast to the somatomedin-like growth factors, FGF and EGF were required continuously in the culture medium during traverse of the entire G1 phase for stimulation of DNA synthesis, and the mitogenic effects of FGF and EGF in cultured chondrocytes were stronger than those of CDF and MSA. Synergistic stimulation of DNA synthesis by CDF or MSA in the presence of FGF or EGF could be observed as long as FGF or EGF was continuously present, even when CDF or MSA was withdrawn after the first 5 h of culture. These findings suggest that, in contrast to FGF and EGF, somatomedin-like growth factors affect an early distinct stage in the G1 phase of chondrocytes.     

Mitogenic stimulation of human breast cancer cells in a growth factor-defined medium: synergistic action of insulin and estrogen

The cooperative action of 17 beta-estradiol (E2) and polypeptide growth factors in stimulating proliferation of human breast cancer cells in vitro was investigated. To prevent background estrogenic stimulation, only phenol red-free media were used. When cultured in media supplemented with steroid-stripped serum in which all polypeptide growth factor activity had been chemically inactivated, MCF7 cells were unable to proliferate and became virtually quiescent. In the additional presence of insulin, epidermal growth factor (EGF), and E2, however, cells proliferated as rapidly as did cells cultured in media supplemented with fetal calf serum. Analysis by DNA flow cytometry showed that in the absence of external growth factors, MCF7 cells became arrested predominantly in the G1/G0 phase of the cell cycle. Upon addition of insulin in combination with EGF and E2, however, cells reentered the cell cycle with a high degree of synchrony. When added alone, E2 induced only slight mitogenic effects under these growth factor-defined conditions. In contrast, this steroid induced optimal proliferation in conventional steroid-stripped serum, which in itself contained considerable mitogenic activity. Insulin (at 10 micrograms/ml) was the most potent stimulator of MCF7 cell proliferation under growth factor-defined conditions, resulting in a more than sixfold increase in cell number after 96 hours. Other growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and EGF had little effect by themselves and only slightly influenced insulin-induced proliferation. At suboptimal concentrations of insulin (10-100 ng/ml), however, strong synergism was observed between E2 and insulin in inducing MCF7 proliferation. Using the CG5 cell line, a highly E2-sensitive MCF7 variant, synergism with E2 was already observed at 1 ng/ml insulin. It is concluded that MCF7 cells require insulin (or insulin-like growth factors) for proliferation. At suboptimal insulin concentrations, E2 acts synergistically with insulin, possibly by inducing autocrine production of polypeptide growth factors by these cells.     

Increased glutathione levels in quiescent, serum-stimulated NRK-49F cells are associated not with a response to growth factors but with nutrient repletion.

Treatment of quiescent cells with serum results concomitantly in an increase in cellular glutathione (GSH) content and growth stimulation. A possible association between the GSH increase and the growth response was examined by studying separately the effects of nutrients and growth factors on the levels of cellular GSH and proliferation of quiescent NRK-49F cells. The addition of fresh medium with 10% calf serum was found to result in both a twofold increase in cellular GSH and growth stimulation (DNA synthesis and cell proliferation). 10% calf serum alone, without fresh medium, stimulated cell growth but failed to cause a comparable increase in cellular GSH. The addition of fresh medium without 10% serum, and of 0.5 mM cysteine and glutamate, resulted in both instances in a marked increase in cellular GSH, but failed to stimulate cell growth. EGF, in contrast, induced a complete mitogenic response but did not increase cellular GSH. Finally, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GSH synthesis, decreased cellular GSH and inhibited EGF-induced DNA synthesis, but these two responses do not, in their dose dependency, correlate. The results obtained thus show that the increase in cellular GSH that occurs in quiescent, serum-stimulated NRK-49F cells is a result of nutrient repletion rather than mitogenic stimulation, and increased GSH levels do not necessarily precede DNA synthesis and mitosis.     

Early stimulation by EGF plus insulin of rRNA, c-fos, and actin mRNA expression: Inhibition by cytochalasin D

Stimulation of membrane ruffling is one of the first events induced by addition of growth factors to quiescent cultures. In order to assess the importance of intact cytoskeleton in induction, by EGF + insulin, of early events such as stimulation of rRNA, c-fos, and actin mRNA expression, we studied the effect of cytochalasin D (CD) on these metabolisms. We observed that CD slightly increased rRNA synthesis in nonstimulated cells; conversely, it decreased rRNA synthesis in cells stimulated by EGF + insulin. The maximal inhibition observed was 60%. The c-fos mRNA was not expressed in control cells and was accumulated in cells stimulated by the mixture of EGF + insulin; this accumulation was inhibited by CD. Actin mRNA was expressed in control cells and its expression was stimulated by EGF + insulin. Addition of CD decreased actin mRNA accumulation in stimulated cells but increased this accumulation in unstimulated cells. Our results, taken together, show that CD specifically affected the stimulation of rRNA and mRNA expression induced by growth factors and suggest that intact cytoskeleton and possibly membrane ruffling favored this stimulation.     

Effect of Na + flux inhibitors on induction of c-fos, c-myc, and ODC genes during cell cycle

The role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride-sensitive Na+/H+ antiport and the bumetanide-sensitive Na+/K+/Cl- cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain-sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na+/K+ pump stimulation was obtained. Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S-phase was completely inhibited. To investigate the mode of cell-cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes--c-fos, c-myc, and ornithin decarboxylase (ODC)--was measured during cell transition through the G1-phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c-fos, c-myc, and ODC mRNAs. These results suggest that stimulation of Na + fluxes by serum growth factors is essential for cell transition into the S-phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c-fos, c-myc, and ODC genes induction.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.     

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I)-stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I)-stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.     

Hepatocyte DNA replication: effect of nutrients and intermediary metabolites

Isolated adult rat liver parenchymal cells maintained in serum-free medium are stimulated by insulin and epidermal growth factor (EGF) to undergo DNA synthesis. Pyruvate, lactate, and, to a lesser extent, several other intermediary metabolites strikingly enhance DNA synthesis both under serum-free culture conditions and in the presence of dialyzed rat serum. High concentrations (2-50 mM) of these low-molecular-weight metabolites are necessary to produce optimal stimulation. Both alanine (greater than 2 mM) and glutamine (greater than 4 mM) are inhibitory under similar conditions. Glucose, although not required for hepatocyte maintenance or stimulation in the presence of insulin and EGF, acts synergistically with pyruvate to enhance DNA synthesis in a complete mixture.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (> 40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin beta1 (HRG-beta1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.