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Endocrinology Vol. 140, No. 12 5626-5634
Copyright © 1999 by The Endocrine Society

ARTICLES

Epidermal Growth Factor and Insulin-Induced Deoxyribonucleic Acid Synthesis in Primary Rat Hepatocytes Is Phosphatidylinositol 3-Kinase Dependent and Dissociated from Protooncogene Induction1

Christian J. Band, Catherine Mounier and Barry I. Posner

Polypeptide Hormone Laboratory and the Departments of Medicine and Physiology, McGill University, Montréal, Québec, Canada

Address all correspondence and requests for reprints to: Dr. Barry I. Posner, Polypeptide Hormone Laboratory, Strathcona Medical Building, 3640 University Street, Room W315, Montréal, Québec, Canada H3A 2B2. E-mail: mc85{at}musica.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhaps through p70s6k activation, regulates DNA synthesis in response to insulin and EGF; 2) the MAPK pathway is not involved in insulin- and EGF-induced DNA synthesis; and 3) p44/42 MAPKs are involved the induction of c-myc mRNA levels, although this induction is not required for DNA synthesis. These studies define two distinct signal transduction pathways that independently mediate growth-related responses in a physiologically relevant, normal cell system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNDERSTANDING the mechanisms that govern cell proliferation is key to understanding both normal growth and the development of malignancy. The signal transduction pathways involved in mediating cell proliferation are being actively investigated in many different cell culture systems. Considerable effort has been focused on identifying the factors that promote hepatocyte proliferation as an example of a normal physiological process (1, 2, 3). Initial studies in partially hepatectomized animals suggested key roles for epidermal growth factor (EGF) and insulin in liver regeneration (4, 5, 6, 7, 8). Studies in primary hepatocyte cultures, where confounding influences of multiple in vivo changes are absent, have established that insulin and EGF are indeed hepatic mitogens (9, 10, 11, 12, 13).

In most established cell lines, phosphatidylinositol 3-kinase (PI3-kinase) has been identified as the critical effector of insulin-mediated mitogenesis (14, 15, 16). In some cells, however, insulin appears to mediate proliferation, primarily through activation of the mitogen-activated protein (MAP) kinase pathway (17, 18, 19, 20). In primary hepatocytes, the ability of insulin to stimulate DNA synthesis has recently been shown to involve the activation of PI3-kinase and p70s6k (13). The role of the MAP kinase signaling pathway in promoting hepatocyte proliferation in response to insulin has not been addressed.

The MAP kinase pathway is generally viewed as the primary effector of the proliferative response of cells to EGF (21, 22, 23), although PI3-kinase has also been implicated (24). A role for PI3-kinase in EGF-mediated DNA synthesis (12) in primary hepatocytes was excluded based on the lack of effect of wortmannin, a specific inhibitor of PI3-kinase (25, 26). The importance of the MAP kinase pathway in EGF-induced DNA synthesis in primary hepatocytes has not been investigated.

In the present study we examined the relative contributions of the activation of PI3-K and downstream events vs. the activation of the MAP kinase pathway in promoting EGF- and insulin-induced DNA synthesis in subconfluent cultures of primary hepatocytes. Using the PI3-kinase inhibitors wortmannin and LY294002 (27), and PD98059, a specific inhibitor of the p44/42 MAP kinase activator MEK1 (28, 29), we demonstrate that PI3-kinase, but not the MAP kinase pathway, is necessary and sufficient to account for both EGF- and insulin-mediated DNA synthesis in these cells. This PI3-kinase-mediated DNA synthesis was independent of hormone-induced augmentations of the growth-related immediate early genes c-fos, c-jun, and c-myc, which are known markers of the G0/G1 transition preceding DNA synthesis (30, 31, 32).

The MAP kinase pathway was required for an augmentation of basal c-myc messenger RNA (mRNA) levels by EGF in subconfluent hepatocytes, consistent with the ascribed role of MAP kinases in regulating the expression of this gene (33, 34, 35). In confluent, growth-arrested, hepatocytes, c-myc mRNA levels were also induced by EGF in a MAP kinase-dependent manner.

Our studies demonstrate that in primary hepatocyte cultures the MAP kinase and PI3-kinase pathways act independently to effect distinct growth-related responses and provide conceptual insights into the regulation of cell proliferation, which may prove to be generally applicable to normal and aberrant growth regulation in a variety of mammalian tissues in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Porcine insulin was a gift from Lilly Research Laboratories (Indianapolis, IN), and mouse EGF was obtained from Collaborative Biomedical Products (Bedford, MA). Myelin basic protein (MBP), protein kinase inhibitor (P-300), and wortmannin were purchased from Sigma Chemical Co. (St. Louis, MO), and LY294002 was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Rapamycin was obtained from Calbiochem (San Diego, CA), and PD98059 was purchased from New England Biolabs, Inc. (Mississauga, Canada). Collagenase was purchased from Worthington Biochemical Corp. (Freehold, NJ). Cell culture medium and antibiotics were obtained from Life Technologies, Inc. (Burlington, Canada), and Vitrogen-100 was obtained from Collagen Corp. (Toronto, Canada). [3H]methylthymidine, [{alpha}-32P]deoxy-CTP, and 125I-labeled goat antirabbit antibody were obtained from ICN Biomedicals, Inc., Canada Ltd. (Mississauga, Canada). ATP was purchased from Boehringer Mannheim (Laval, Canada), and [{gamma}-32P]ATP was purchased from NEN Life Science Products-DuPont (Wilmington, DE). p44/42 MAP kinase (C-16) and p70s6k (C-18) antibodies and p70s6k peptide substrate (RRRLSSLRA) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Protein A-Sepharose was obtained from Pharmacia Biotech (Montreal, Canada). The PhosphoPlus p44/42 MAP kinase (Thr202/Tyr204) Antibody Kit was purchased from New England Biolabs, Inc. (Beverly, MA). Immobilon-P transfer membranes were obtained from Millipore Corp. Canada Ltd. (Mississauga, Canada). All other reagents were obtained from Sigma Chemical Co. and were of the highest grade available.

Cell culture
Primary hepatocytes, isolated from 160- to 180-g male Sprague Dawley rats (Charles River Laboratories, Inc., St. Constant, Canada) by in situ liver perfusion with collagenase (the animal protocol was approved by the animal care committee of McGill University and given protocol no. 4110), were plated on a collagen matrix (Vitrogen-100). Subconfluent and confluent cultures were prepared by seeding 1 x 106 and 3 x 106 cells, respectively, onto 9.6 cm2 six-well plates (Corning, Costar, Cambridge, MA) or 5 x 106 and 1.5 x 107 cells, respectively, onto 78-cm2 culture dishes (Starstedt Canada, St. Laurent, Canada). Cells were bathed for 24 h in seeding medium (DMEM/Ham’s F-12 containing 10% FBS, 10 mM HEPES, 20 mM NaHCO3, 500 IU/ml penicillin, and 500 µg/ml streptomycin) and then for 24 h in serum-free medium (SFM) that differed from the seeding medium in that it lacked FBS and contained 1.25 µg/ml fungizone, 0.4 mM ornithine, 2.25 µg/ml L-lactic acid, 2.5 x 10-8 M selenium, and 1 x 10-8 M ethanolamine. SFM was renewed before the addition of [3H]thymidine, hormones, and inhibitors, as described below.

[3H]Thymidine incorporation assay
Subconfluent or confluent cultures were plated on 9.6-cm2 six-well plates in serum-containing medium for 24 h, and then in serum- and growth factor-free medium for an additional 24 h. Insulin or EGF and [3H]thymidine (5 µCi/ml) were added to cells preincubated for 30 min with or without wortmannin, LY294002, rapamycin, PD98059, or dimethylsulfoxide (DMSO) vehicle. The concentrations of the test agents are specified in the figure legends. After an 18-h incubation, cells were rinsed twice with 3 ml cold PBS, incubated for 15 min at 4 C in 10% trichloroacetic acid, solubilized at room temperature in 1 ml 1 N NaOH, and then transferred to scintillation vials and counted for 3H.

RNA extraction, dot blot hybridization, and protooncogene mRNA quantitation
c-fos, c-jun, and c-myc complementary DNAs (cDNAs) were provided by Dr. John Bergeron (Department of Anatomy, McGill University, Montreal, Canada). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was described previously (36). Total RNA was isolated from hepatocytes cultured on 72-cm2 petri dishes according to Chomczynski’s method (37). Dot blot analyses of 6.5 µg total RNA were performed on Hybond-N nylon membranes in a dot-blot manifold (Bio-Rad Laboratories, Inc., Richmond, CA), according to the manufacturer’s protocol. RNA was fixed to the membranes by UV cross-linking and hybridized sequentially, with intermittent stripping, with c-fos, c-jun, c-myc, and GAPDH cDNA probes labeled with [{alpha}-32P]deoxy-CTP to a specific activity of 109 dpm/µg using T7 QuickPrime (Pharmacia Biotech). Membrane hybridization, washing, and stripping conditions were described previously (36). Blots were exposed to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) at -70 C for different durations and were quantitated using a GS-700 Imaging Densitometer (Bio-Rad Laboratories, Inc., Hercules, CA). Ratios of the amount of protooncogene mRNA and GAPDH mRNA in each dot blot were expressed as a percentage of their ratios in appropriate controls, which were normalized to 100% (see figure legends).

Preparation of cell lysates for enzyme assays and Western blot analysis
After treatment with the test agents described in the figure legends, hepatocytes were rinsed twice with cold PBS (pH 7.4) and lysed at 4 C by adding 1 ml/well lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1.5 mM MgCl2, 1 mM EGTA, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10% glycerol, and 1% Triton X-100]. Lysates were centrifuged at 10,000 x g for 20 min, and protein concentrations in the resulting supernatants were determined by the method of Bradford using BSA as a standard (38).

p44/42 MAP kinase activity assay
The activity of p44/42 MAP kinase was analyzed using two different methods. The in vitro kinase assay using MBP as substrate was described previously (39), with slight modifications. Briefly, cell lysates (1 mg protein) were incubated with mild agitation for 90 min at 4 C with 5 µl p444/42 MAP kinase antiserum preadsorbed to protein A-Sepharose beads. This antibody recognizes p42 and p44 isoforms of MAP kinase. The beads were washed three times with lysis buffer and twice with MAP kinase assay buffer [50 mM HEPES (pH 7.4), 5 mM magnesium acetate, 2 mM dithiothreitol, 1 mM EGTA, and 0.2 mM sodium orthovanadate]. The phosphorylation of MBP was assayed by resuspending the beads in a total final volume of 100 µl MAP kinase assay buffer containing 25 µg/ml MBP, 50 µm ATP, and 1 µCi [{gamma}-32P]ATP. Reactions, initiated upon addition of [{gamma}-32P]ATP, were carried out at 30 C for 30 min and terminated by the addition of 25 µl 5 x Laemmli sample buffer and boiling for 5 min. Samples were subsequently subjected to SDS-PAGE on 12.5% gels, after which gels were incubated for 3 h in 5% acetic acid-17% methanol-78% H2O, dried under vacuum, and exposed to x-ray film. p44/42 MAP kinase activity was also assessed by Western blot analysis using the PhosphoPlus Antibody Kit according to the manufacturer’s protocol. This method employs a phospho-p44/42 MAP kinase antibody that reacts specifically with the activated form of p44/42 MAP kinase.

p70s6k activity assay
Hepatocyte lysates (1 mg protein) with 2 µg p70s6k antibody (C-18, Santa Cruz Biotechnology, Inc.) preadsorbed to protein A-Sepharose beads were gently agitated for 90 min at 4 C. Immune complexes were washed three times with lysis buffer and twice with p70s6k assay buffer, which was identical to the MAP kinase assay buffer except that it contained protein kinase inhibitor (4 µm final concentration). The beads were resuspended in a final volume of 100 µl p70s6k buffer containing 500 ng S6 peptide RRRLSSLRA (Santa Cruz Biotechnology, Inc.), 50 µm ATP, and 1 µCi [{gamma}-32P]ATP. Reactions, initiated by the addition of ATP, were carried out at 30 C for 20 min and were terminated by the addition of 10 µl 88% formic acid. The reaction products were spotted on phosphocellulose P-81 filters (Whatman, Milford, MA), which were washed four times for 15 min each time with 500 ml 1% phosphoric acid, twice with distilled water, and once in ethanol and were counted in scintillation fluid (40).

MAP kinase Western blot
After the addition of Laemmli buffer, lysates containing 30 µg protein were boiled for 5 min and subjected to SDS-PAGE under reducing conditions before electrophoretic transfer of proteins onto Immobilon-P membranes. The membranes were incubated overnight at 4 C in blocking solution, which consisted of TNT buffer [300 mM NaCl, 10 mM Tris (pH 7.4), and 0.05% Tween-20] containing 5% powdered milk, and then for 2 h at room temperature in blocking solution containing p44/42 antiserum (1:2500 dilution). The blots were washed three times for 10 min each time in 50 ml TNT buffer containing 0.5% milk, incubated for 1 h at room temperature in blocking solution with [125I]goat antirabbit antibody antibody (700,00 cpm/electrophoretic lane transferred), and washed three times as described above. The blots were air-dried and exposed to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) at -80 C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent effects of EGF and insulin on [3H]thymidine incorporation into DNA in subconfluent and confluent hepatocyte cultures
EGF and insulin have been shown to promote DNA synthesis and mitosis in adult primary rat hepatocytes under a variety of culture conditions (9, 10, 11, 12, 13). The dose-dependent effects of these hormones on DNA synthesis in serum-deprived hepatocytes, shown in Fig. 1Go, are consistent with receptor-specific mediated responses. In subconfluent cultures, insulin and EGF maximally stimulated [3H]thymidine incorporation into DNA at 100 nM and 10 nM, respectively. In subsequent studies insulin and EGF were used at a concentration of 100 nM. The degree of stimulation at these doses varied, depending on the hepatocyte preparation, from 1.5- to 3.5-fold over nonstimulated control values (compare Figs. 1Go and 2aGo). As expected, in confluent hepatocytes basal DNA synthesis was low, and no statistically significant stimulation was seen upon addition of EGF. For insulin, we observed stimulation of [3H]thymidine incorporation into DNA (Fig. 1bGo), but the level of stimulation was less than that observed in subconfluent cells. Similar observations were made by Kimura and Ogihara (13), who showed a density-dependent reduction of EGF-induced DNA synthesis with total inhibition in confluent cells, but no such density effect on insulin action. Basal DNA synthesis (Fig. 1Go, arrows) was 4-fold higher in subconfluent vs. confluent cells, demonstrating the former’s capacity for growth autoregulation. A comparison of basal DNA synthesis in confluent cells and maximally stimulated DNA synthesis in subconfluent cells revealed a 9-fold difference, presumably reflecting the proliferative potential of the hepatocytes in our culture system.



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  		Figure 1. Dose response to insulin and EGF on stimulation of DNA synthesis in hepatocytes. Serum-starved subconfluent (left panel) and confluent (right panel) hepatocytes were incubated for 18 h in SFM containing 5 µCi [3H]methylthymidine with the indicated concentrations of insulin (solid circles) and EGF (open circles). Incorporation of [3H]thymidine into DNA was determined as described in Materials and Methods and was normalized to cell number. The results are expressed as the mean ± SD from three separate experiments.

 


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  		Figure 2. Effects of wortmannin, LY294002, and rapamycin on insulin- and EGF-induced DNA synthesis and p70s6k activation. A, Serum-starved subconfluent hepatocytes were incubated for 30 min with DMSO carrier, wortmannin, LY294002, or rapamycin before an 18-h incubation in SFM containing 5 µCi [3H]methylthymidine with or without 100 nM insulin or EGF. Incorporation of [3H]thymidine into DNA was determined as described in Materials and Methods. Results are expressed as the mean ± SD from three separate experiments. B, Lysates were obtained from serum-starved subconfluent hepatocytes stimulated for 10 min with insulin or EGF with prior incubation for 30 min with DMSO carrier, wortmannin, or rapamycin. Immunoprecipitates obtained with a selective antibody to p70s6k were assayed for their ability to phosphorylate the ribosomal S6 kinase substrate RRRLSSLRA in the presence of [{gamma}-32P]ATP. The results, expressed as 32P incorporated into substrate (counts per min), are the mean ± SD of measurement on samples from three separate plates.

 
PI3-kinase and mTOR are involved in EGF and insulin-induced DNA synthesis in subconfluent primary rat hepatocytes
We evaluated the effects of two specific and mechanistically distinct PI3-kinase inhibitors, wortmannin and LY2940020 (25, 27), and that of rapamycin, which inhibits p70s6k activation (41, 42), on [3H]thymidine incorporation into DNA. PI3-kinase activated by growth factors, including insulin and EGF, has been implicated in DNA synthesis in various cell lines (14, 15, 16, 24, 43, 44). We show that 100 nM wortmannin almost fully reversed and that 50 µM LY294002 completely inhibited insulin-stimulated DNA synthesis (Fig. 2aGo), demonstrating a critical role for PI3-kinase in insulin-induced proliferation in primary hepatocytes. Of particular interest, we found that EGF-induced DNA synthesis was equally sensitive to wortmannin and LY294002 (Fig. 2aGo). This is the first report ascribing a role for PI3-kinase in DNA synthesis induced by EGF in primary cell culture.

The ribosomal protein S6 kinase, p70s6k, is activated by PI3-kinase in response to most growth factors by mechanisms that have not been fully elucidated (41, 45, 46, 47, 48). We have recently shown a requirement for PI3-kinase of p70s6k activation by insulin in primary rat hepatocytes (36). Inhibition of p70s6k antagonizes the transition of cells through the G1/S phase of the cell cycle (49, 50). The immunosuppressant agent rapamycin is a potent inhibitor of p70s6k at the level of the mammalian target of rapamycin mTOR (FRAP/RAFT) (51, 52). We found that 50 nM rapamycin powerfully reduced constitutive, insulin-induced, and EGF-induced DNA synthesis to the same absolute level, which was approximately 50% that of unstimulated hepatocytes (Fig. 2aGo). Wortmannin and LY294002 also reduced basal DNA synthesis (Fig. 2aGo). Thus, PI3-kinase, mTOR, and probably p70s6k, in addition to being involved in EGF- and insulin-stimulated proliferation, appear to regulate constitutive DNA synthesis in hepatocytes. Overall, we found that for any given treatment DNA synthesis (Fig. 2aGo) correlated well with p70s6k activity (Fig. 2b).

Increased MAP kinase activity is not involved in insulin and EGF-induced DNA synthesis
Insulin and EGF treatment of cells activates Ras, resulting in activation of the MAP kinase signaling pathway (53). Dominant negative mutants of Ras and antisense oligonucleotides to Raf1, MEK1, and MAP kinase block insulin- and EGF-induced DNA synthesis in established cell lines (18, 19, 20, 21, 53). Rodriguez-Viciana et al. (54) provided evidence that PI3-kinase is a downstream target of Ras. On the other hand, activation of the Ras/Raf1/MEK1/MAP kinase signaling pathway by expression of a constitutively active catalytic subunit of PI3-kinase, p110, suggests that Ras is an effector of PI3-kinase (55). We described an inhibitory effect of wortmannin on insulin-induced MAP kinase activation in primary rat hepatocytes (36). Thus, the inhibitory effects of wortmannin on DNA synthesis (Fig. 2aGo) may result from decreased MAP kinase activity. We assessed the impact of inhibiting the most proximal activator of MAP kinase, MEK1, with the selective MEK antagonist PD98059 (28, 29), in our proliferation assay. PD98059 had no statistically significant effect on basal, insulin-induced, or EGF-induced DNA synthesis (Fig. 3aGo) even when tested at a dose of 60 µM (data not shown). As shown in Fig. 3bGo, PD98059 completely suppressed MAP kinase activation by both hormones. This inhibition persisted for the full 18-h incubation with [3H]thymidine (data not shown). Moreover, the stronger stimulation of MAP kinase activity effected by EGF compared with insulin (Figs. 3bGo and 6aGo) was not paralleled by a greater effect of the former on DNA synthesis. Regardless of the interrelationship between PI3-kinase and Ras, we conclude that neither MEK1 nor p44/42 MAP kinase is involved in the stimulatory effect of insulin and EGF on DNA synthesis in primary rat hepatocytes.



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  		Figure 3. Effect of PD98059 on insulin- and EGF-induced DNA synthesis and p44/42 MAP kinase activation. A, Serum-starved subconfluent hepatocytes were incubated for 30 min with DMSO carrier or PD98059 before an 18-h incubation in SFM containing 5 µCi [3H]methylthymidine with or without 100 nM insulin or EGF. Incorporation of [3H]thymidine into DNA was determined as described in Materials and Methods. Results are expressed as the mean ± SD from three separate experiments. B, p44/42 MAP kinase immunoprecipitates were prepared from lysates obtained from subconfluent hepatocytes, untreated (basal) or treated for 5 min with 100 nM insulin or EGF with prior incubation for 30 min with DMSO carrier or PD98059. MAP kinase activity was assayed as described in Materials and Methods. Shown is a representative autoradiograph of 32P-phosphorylated MBP substrate resolved on a 12.5% polyacrylamide gel.

 


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  		Figure 6. EGF-induced MAP kinase phosphorylation and p44/42 MAP kinase protein levels in subconfluent and confluent hepatocytes. A, Western blot of phospho-p44/42 MAP kinases from lysates of subconfluent and confluent hepatocytes that were untreated (basal) or treated for 5 min with 100 nM EGF with or without 30 µM PD98059. Shown is a representative autoradiograph of phosphorylated, active forms of p44/42 MAP kinases. B, Total cell lysates from unstimulated subconfluent (lane 1) and confluent (lane 2) hepatocytes were subjected to SDS-PAGE and Western blotted with anti-p44/42 MAP kinase antibody as described in Materials and Methods. Indicated are bands corresponding to the p44 and p42 MAP kinase isoforms.

 
Effect of insulin and EGF on protooncogene mRNA induction
Proliferation of hepatocytes has been correlated with augmented levels of c-fos, c-jun, and c-myc mRNAs (56, 57). EGF has been shown to augment c-fos and c-myc mRNA levels in primary hepatocyte cultures (56), suggesting a role for these protooncogenes in mitogenesis effected by this hormone. Insulin activates c-fos transcription in numerous cell lines (58, 59) and both p21ras (20) and PI3-kinase (14, 60) have been implicated in this response. In 12- to 13-day-old quiescent primary hepatocytes, basal c-fos mRNA levels are undetectable and are not augmented by insulin (56). We evaluated levels of c-fos, c-jun, and c-myc mRNAs to determine whether the proliferative effect of EGF and insulin was consequent to their induction. In subconfluent hepatocytes, the expression of neither c-fos, c-jun, nor c-myc mRNA was altered by insulin at 1 h (Fig. 4Go) or at 5 min, 15 min, 30 min, 6 h, and 10 h (data not shown). EGF induced an increase in c-myc mRNA that was apparent by 30 min (data not shown), peaked at 1 h (Fig. 4Go), and returned to basal levels between 6–10 h (data not shown). Thus, insulin-stimulated DNA synthesis does not require an augmentation of c-fos, c-jun, and c-myc mRNAs, whereas EGF-induced DNA synthesis may involve c-myc. However, confluent hepatocytes that did not synthesize DNA (Fig. 1Go, inset) expressed similar basal levels of c-fos, c-jun, and c-myc mRNAs as their subconfluent counterparts (data not shown) and responded to EGF with the same fold induction of c-myc mRNA (Fig. 5Go). Thus, c-myc mRNA induction can be dissociated from, does not precede, and is not subsequent to proliferation induced by EGF, implying that EGF signaling to c-myc is different from that which mediates DNA synthesis.



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  		Figure 4. Effects of insulin and EGF on protooncogene mRNA expression in subconfluent primary hepatocytes. Serum-starved subconfluent hepatocytes were incubated for 1 h with or without 100 nM insulin or EGF. Total RNA was extracted and subjected to dot blot analysis using 32P-labeled probes specific for c-fos, c-jun, c-myc, and GAPDH mRNAs as described in Materials and Methods. The ratios of the densitometric reading of the dot blots of protooncogene mRNA and corresponding GAPDH mRNA are expressed as a percentage of that in control cells (basal), which were normalized to 100%. Results are expressed as the mean ± SD from three separate experiments.

 


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  		Figure 5. Effect of PD98059 on EGF-induced expression of c-myc mRNA in subconfluent and confluent hepatocytes. Serum-starved subconfluent and confluent hepatocytes were incubated for 1 h with or without 100 nM EGF after a 30-min preincubation with DMSO carrier or 30 µM PD98059. Total RNA was extracted and subjected to dot blot analysis using 32P-labeled probes specific for c-myc and GAPDH mRNAs as described in Materials and Methods. The ratios of the densitometric reading of the dot blots of protooncogene mRNA and corresponding GAPDH mRNA are expressed as a percentage of that in control cells (basal), which were normalized to 100%. Results are expressed as the mean ± SE from three separate experiments.

 
p44/42 MAP kinases and EGF induction of c-myc mRNA
Sustained MAP kinase activation favors its translocation to the nucleus (reviewed in Ref. 61), where it may potentially lead to activation of c-myc transcription (34, 35). As noted above, at saturating doses, EGF more powerfully stimulated MAP kinase activity than insulin (Fig. 3bGo). In addition, kinetic studies revealed that in response to EGF, MAP kinase activity was sustained for up to 60 min, whereas insulin induction of MAP kinase was terminated between 15–30 min (data not shown). We tested the effect of PD98059 on EGF-induced c-myc mRNA levels and found that it reversed the stimulatory effect of EGF in subconfluent and confluent hepatocytes (Fig. 5Go), entirely consistent with a role of MAP kinase in inducing c-myc mRNA. The ability of PD98059 to reverse c-myc mRNA induction without affecting DNA synthesis (Fig. 3aGo) clearly demonstrates that these two events are mediated by distinct signaling pathways. In confluent hepatocytes, EGF activation of p44/42 MAP kinase (Fig. 6a) was seen despite barely detectable levels of MAP kinase protein expression in lysates from these cells (Fig. 6bGo), suggesting efficient coupling between MAP kinases and c-myc mRNA induction.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hepatocytes in the normal adult liver are arrested in G0, but acquire a remarkable capacity to proliferate after partial hepatectomy or other treatments resulting in liver cell loss (3, 62). Important roles have been established for insulin and EGF in compensatory liver growth (6, 7, 8, 63, 64). Among the earliest events observed after partial hepatectomy is the rapid and sequential induction of immediate early genes for c-fos, c-jun, and c-myc, all of which are undetectable in normal adult rat liver (8, 31, 56, 65, 66). The induction of these genes is a hallmark of the G0/G1 transition of the cell cycle and is viewed as necessary to render hepatocytes competent to respond to the proliferative effects of hormonal factors (1, 8). Indeed, neither EGF nor insulin induces liver DNA synthesis when administered to normal rats, whereas EGF mediates hepatic DNA synthesis when administered after partial hepatectomy or chemically induced liver cell necrosis (8, 64) in a manner proportional to the level of preexisting c-myc mRNA (8). The pattern of protooncogene expression seen after partial hepatectomy is stimulated during the isolation and culture of primary rat hepatocytes (56). Hepatocytes cultured on rat tail collagen express elevated levels of c-jun and c-myc mRNAs for up to 3 days in culture, during the course of which they synthesize DNA in response to EGF (57). We have used a system of primary rat hepatocytes cultured on a type I collagen matrix to study in detail the signal transduction pathways underlying EGF- and insulin-mediated DNA synthesis.

The first main finding of our study is the novel demonstration that PI3-kinase is both necessary and sufficient to account for EGF-mediated DNA synthesis in subconfluent primary hepatocytes. We also confirm and extend the recent observation that PI3-kinase is involved in insulin-mediated DNA synthesis in hepatocyte cultures (13). EGF and insulin comparably stimulated antiphosphotyrosine-immunoprecipitable PI3-kinase activity (data not shown), and p70s6k activity. In addition, p70s6k activity correlated well with DNA synthesis and was inhibited by wortmannin, thus supporting its role as a major downstream target mediating the proliferative effects of PI3-kinase induced by EGF and insulin. This is consistent with the established role of p70s6k in the G1/S phase transition (49, 50). In contrast to our observations, Kimura et al. failed to demonstrate an inhibitory effect of wortmannin on EGF-induced DNA synthesis in primary rat hepatocytes even though they observed that rapamycin was inhibitory (12). Our data, however, are based upon the effects of two mechanistically distinct PI3-kinase inhibitors, both of which abrogated EGF-induced DNA synthesis. It is noteworthy that wortmannin did not suppress EGF- and insulin-mediated DNA synthesis to the same extent as LY294002. At the doses used in our study, both agents are equally potent inhibitors of PI3-kinase activity (data not shown). Previous work has indicated that wortmannin (0.1–1 µM) and LY294002 (1–30 µM) are capable of maximally inhibiting the autokinase activity of mTOR (67). In this study we used a 100-nM dose of wortmannin and a 50-µM dose of LY294002. Therefore, the greater inhibition of both DNA synthesis and p70 S6 kinase activity by LY294002 compared with wortmannin could reflect the inhibition of mTOR and its role in transducing signals for DNA synthesis. It is of interest to note that at these concentrations, wortmannin is also capable of inhibiting other kinases, such as PI4-kinase (68). However, the expression of dominant negative p85 in rat primary hepatocytes mimics the effect of the pharmaceuticals inhibitors, thus probably excluding such a kinase in insulin- and EGF-induced DNA synthesis (Mei, K., C. Mounier, J. Wu, and B. I. Posner, manuscript in preparation).

We have previously demonstrated that PI3-kinase is upstream of p44/42 MAP kinases in the insulin signaling pathway in primary hepatocytes (36). It is conceivable that in an analogous manner, EGF-activated PI3-kinase is upstream of MAP kinases. However, the lack of an effect of PD98059 on DNA synthesis induced by insulin or EGF demonstrated that MEK1 and p44/42 MAP kinases do not contribute to the overall realization of this response. Moreover, the greater stimulation of p44/42 MAP kinases effected by EGF compared with insulin was not paralleled by a greater proliferative response.

PI3-kinase and/or its lipid products may activate several downstream targets other than p70s6k (69). These include certain isoforms of protein kinase C (70, 71), whose impact on DNA synthesis was not addressed in the present study. Isoforms of protein kinase C that activate Raf1 (72) would not be expected to contribute to EGF- and insulin-stimulated DNA synthesis unless a signal from Raf1 bifurcates upstream of MEK1.

It is interesting to note that DNA synthesis in subconfluent hepatocytes is considerable in the absence of added hormone (Fig. 1Go, compare subconfluent vs. confluent cells), and that it is antagonized by wortmannin, LY294002, and rapamycin, but not PD98059. This raises the possibility that progression into S phase may be effected by stimulatory autocrine factors (73) that operate through the same signaling pathway as EGF and insulin or by cell-cell and/or cell-matrix interactions, which in other cells have been shown to activate PI3-kinase and p70s6k (74). This result contrasts with the recently described restriction point in mid to late G1, beyond which hepatocytes could not progress without mitogenic stimulation (75), and presumably reflects different culture conditions, particularly our use of a collagen matrix. On the basis of previous work (75), it is likely that insulin and EGF act predominantly by increasing the population of hepatocytes undergoing the G1/S transition, rather than by increasing the rate of DNA synthesis in a fixed pool of cells. This is emphasized by the work of Kimura and Ogihara (13), who described a good correlation between [3H]thymidine incorporation and nuclear labeling in rat primary hepatocytes stimulated by insulin and EGF. Also favoring this possibility is the finding that rapamycin, which arrests cells before, but not after, their entry into S phase (76), was extremely powerful in inhibiting insulin- and EGF-mediated DNA synthesis.

A second main finding was derived from our studies on immediate early gene expression. Unlike normal adult rat liver in which protooncogene mRNA levels are undetectable, hepatocytes cultured on a collagen matrix expressed substantial basal levels of c-fos, c-jun, and c-myc mRNAs, which we believe were sufficient to support DNA synthesis, as the latter was not contingent upon further augmentations of these mRNA species. The EGF-induced increase in c-myc mRNA levels was of considerable interest because of the recognized role of c-myc as a necessary component for entry of cells into S phase (77, 78, 79) (see below).

In line with the evidence supporting a role for the MAP kinase pathway (80), and particularly p44/42 MAP kinases, in the induction of c-myc gene transcription (33, 34, 35), the EGF-mediated augmentation of c-myc mRNA in subconfluent and confluent hepatocyte cultures was completely abrogated by PD98059, a highly specific inhibitor of MEK1 (28, 29), for which p44/42 MAP kinases are the only known in vivo substrates (81). The induction of c-myc mRNA can thus be entirely explained by activation of p44/42 MAP kinases through EGF activation of MEK1. It is, however, possible that as yet uncharacterized cytosolic substrates of MEK1 exist, which upon activation could signal the c-myc gene. Alternatively, MEK1 could activate membrane-associated MAP kinases within discrete subcellular compartments. Such a mechanism has been proposed as the basis for Golgi fragmentation during mitosis (82). The recent demonstration that MEK1 translocates to the nucleus (83) raises the possibility that it may itself, directly or indirectly, impact on gene regulation. The kinetics of activation of the MAP kinase pathway are important determinants of cellular responses (61). The ability of EGF, but not insulin, to induce c-myc mRNA levels is consistent with its more powerful and sustained level of p44/42 MAP kinase activation (data not shown).

Pledger et al. defined two sequential phases of the cell cycle in mammalian fibroblasts, termed competence and progression, based on the observation that platelet-derived growth factor, by itself, could not stimulate DNA synthesis, but rendered cells competent to progress into S phase in response to progression factors present in plasma (84). Kaczmarek et al. showed that microinjection of c-Myc protein into fibroblasts mimicked the effect of platelet-derived growth factor and established c-Myc as a competence factor (76). Earlier studies in hepatocytes cultured directly onto plastic petri dishes showed that EGF was a complete mitogen (85), whereas insulin induced DNA synthesis only after prior exposure of cells to EGF (85, 86). In the present studies further augmentations of c-myc mRNA levels were not required for DNA synthesis. However, the observation that EGF, but not insulin, augmented c-myc mRNA is consistent with EGF being a competence and a progression factor in primary rat hepatocytes, and with insulin acting as a progression factor in hepatocytes rendered competent by basal expression of c-myc. An understanding of the mechanisms that underlie growth factor-mediated mitogenic responses in their target tissues requires an assessment of immediate early gene expression in parallel with growth factor effects on DNA synthesis.

The present studies provide a rationalization of the competence-progression model of Pledger et al. (83) in terms of signal transduction; the MAP kinase pathway, through the induction of c-myc mRNA, confers competence, and the PI3-kinase pathway promotes G1/S phase progression and DNA synthesis. The similarities in protooncogene mRNA expression in primary hepatocytes cultured on a collagen matrix and those seen in vivo after partial hepatectomy support a role for insulin as an important growth factor during compensatory liver growth.

In summary, we have delineated two distinct signaling pathways with important roles in DNA synthesis in a physiologically relevant, nontransformed cell system: 1) the MAP kinase pathway, which regulates c-myc mRNA expression; and 2) the PI3-kinase/p70s6k pathway, which mediates DNA synthesis. It will be of interest to see whether similar results will be obtained in other cell systems.


   Acknowledgments
 
The authors thank Dr. Jiong Woo for his insightful discussion.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council and the NCI of Canada. Back

Received January 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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