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Epidermal growth factor–induced activation of the insulin-like growth factor I receptor in rat hepatocytes
Epidermal Growth Factor–Induced Activation of the Insulin-Like Growth Factor I Receptor in Rat Hepatocytes Hazem Hallak, Giesla Moehren, Jei Tang, Mohamad Kaou, Mouhamad Addas, Jan B. Hoek, and Raphael Rubin Insulin-like growth factor I (IGF-I) plays a critical role in the induction of cell cycle progression and survival in many cell types. However, there is minimal IGF-I binding to hepatocytes, and a role for IGF-I in hepatocyte signaling has not been elucidated. The dynamics of IGF-I receptor (IGF-IR) activation were examined in freshly isolated rat hepatocytes. IGF-I did not activate the IGF-IR. However, des(1-3)IGF-I, which weakly binds IGF binding protein-3 (IGFBP-3), induced IGF-IR phosphorylation. IGFBP-3 surface coating was identified by confocal immunofluorescence microscopy. In contrast with the inactivity of IGF-I, epidermal growth factor (EGF) induced the tyrosine phosphorylation of the IGF-IR in parallel with EGF receptor phosphorylation. Transactivation of the IGF-IR by EGF was inhibited by tyrphostin I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR. Src kinase inhibitors pyrazolopyrimidine PP-1 and PP-2 inhibited transactivation of the IGF-IR by EGF. EGF stimulated the tyrosine phosphorylation of Src, and induced its association with the IGF-IR. EGF-induced phosphorylations of insulinrelated substrate (IRS)-1, IRS-2, Akt, and p42/44 mitogen-activated protein kinases (MAPKs) were inhibited variably by I-Ome-AG538. In conclusion, the data show an EGF- and Src-mediated transactivation pathway for IGF-IR activation in hepatocytes, and indicate a role for the IGF-IR in hepatocyte intracellular signaling. The findings also show a role for IGFBP-3 in the inhibition of IGF-I signaling in hepatocytes. (HEPATOLOGY 2002;36:1509-1518.)
I
nsulin-like growth factor I (IGF-I) is a critical regulator of cell proliferation and survival.1,2 For many cell types, IGF-I induces mitogenesis, and also is a direct inhibitor of apoptosis. IGF-I signaling is initiated by high-affinity binding to the IGF-I receptor (IGF-IR). The IGF-IR exists as an ␣2-2 heterodimer. The extracellular ligand-binding domain is constituted on the ␣ chain, and the intracellular  domain contains a classic tyrosine kinase that is over 80% homologous to the insu-
Abbreviations: IGF-I, insulin-like growth factor I; IFT-IR, insulin-like growth factor I receptor; IRS, insulin-related substrates; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Ig, immunoglobulin; PBS, phosphate-buffered saline; IGFBP-3, insulin-like growth factor binding protein-3; IR, insulin receptor. From the Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA. Received June 20, 2002; accepted September 15, 2002. Supported by National Institutes of Health grants AA09976, AA07186, and AA08714. Address reprint requests to: Raphael Rubin, M.D., Department of Pathology, Anatomy and Cell Biology, 1020 Locust St., Room 226 Alumni Hall, Philadelphia, PA 19107. E-mail: [email protected]; fax: 215-955-8703. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3606-0027$35.00/0 doi:10.1053/jhep.2002.37138
lin receptor tyrosine kinase. On ligand binding, the receptor undergoes autophosphorylation within the tyrosine kinase domain. Other key tyrosines that are important for downstream signaling are located in the juxtamembrane region and within the carboxyterminal domain. The roles of the respective tyrosines in cell proliferation, survival, and transformation have been dissected by means of sitedirected mutagenesis, and have been reviewed elsewhere.3 Intracellular signaling by the IGF-IR is exceedingly similar to that of the insulin receptor.2,4 After IGF-I binding, the IGF-IR undergoes rapid tyrosine autophosphorylation, and then phosphorylates key signaling molecules including a family of insulin-related substrates (IRS) and Shc. Tyrosine-phosphorylated IRS-1, IRS-2, and Shc bind several molecules that bear Src homology 2 adaptor sites such as Grb2, which leads to activation of p21ras and the mitogen-activated protein kinases (MAPK). The antiapoptotic properties of the IGF-IR principally are mediated by IRS-1 binding to the p85 subunit of phosphatidylinositol 3-kinase, and the subsequent activation of Akt/PKB.5,6 Liver regeneration is a process that is controlled tightly by an array of cytokines and growth factors.7 Factors that 1509
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have been identified that prime hepatocytes for mitogenesis include tumor necrosis factor ␣ and interleukin-6. Hepatic mitogens that induce G1-S phase transition include hepatocyte growth factor, transforming growth factor , and epidermal growth factor (EGF). The mitogenic action of these growth factors is enhanced by comitogens including insulin, glucagon, norepinephrine, thyroid hormone, parathormone, prolactin, and estrogen. In contrast with most cell types, a definitive role for IGF-I in hepatocyte growth has not been delineated. The liver is the major source of circulating IGF-I8 but there is negligible IGF-I binding to hepatocytes.9-12 IGF-I has been reported to induce modest metabolic effects and only slight enhancement of DNA synthesis in cultured hepatocytes,13-15 which likely is mediated by IGF-I binding to insulin receptors.14,16,17 IGF-I binding to rat liver plasma membranes and isolated hepatocytes increases after several days of liver regeneration.9,10 In this study, we have assessed the potential signaling role of the IGF-IR in rat hepatocytes. We confirm the inactivity of IGF-I toward the IGF-IR in freshly isolated rat hepatocytes and identify an inhibitory role for surfaceassociated IGF-I binding protein-3. In contrast, EGF induces the rapid tyrosine phosphorylation of the IGF-IR, a process that is dependent on Src kinase activation. We further show a role for the transactivated IGF-IR in the phosphorylation of key downstream signaling mediators.
Materials and Methods Materials. Human recombinant IGF-I was purchased from Calbiochem (San Diego, CA). EGF was purchased from Sigma (St. Louis, MO). Des(1-3)-IGF-I was obtained from Gropep (Adelaide, Australia). Anti–IGFIR -subunit rabbit polyclonal antibodies and antiphosphotyrosine (PY99) mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGF receptor (EGFR) antibodies and the EGFR antagonist AG1478 were obtained from Calbiochem. Anti–IRS-1 and anti–IRS-2 antibodies (rabbit, polyclonal immunoglobulin [Ig]G) were obtained from UBI (Lake Placid, NY). Anti-MAP kinase antibodies, anti-phosphotyrosine and anti-phosphothreonine MAP kinase (E10) antibodies, horseradish peroxidase– conjugated goat anti-rabbit IgG, horse anti-mouse IgG antibodies, and signal-enhanced chemiluminescence reagents were obtained from New England Biolabs (Beverly, MA). Anti– c-Src antibody (Src-2; sc-18) was purchased from Santa Cruz Biotechnology, and anti–phospho-Src antibodies (pY215) were purchased from Biosource (Camarillo, CA). Anti-Akt antibodies were from Cell Signaling (Beverly, MA). Pyrazolopyrimidines PP-1, PP-2, and
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PP-3 were purchased from Biomol (Plymouth Meeting, PA). Tyrphostin I-Ome-AG538 was a generous gift from Dr. A. Levitzki, Hebrew University, Jerusalem. All other chemicals and biochemicals were of the highest purity commercially available. Hepatocyte Preparation and Culture Conditions. Hepatocytes were isolated by collagenase perfusion as described previously.18 Cells were washed 4 times to remove nonparenchymal cells, which was verified by visual inspection. Freshly isolated hepatocytes were cultured for 2 hours in serum-free Williams medium E at 37°C, 5% CO2, before the addition of growth factors. HepG2 cells were purchased from ATCC (Manassas, VA), and were grown in minimum essential Eagle medium with 2 mmol/L L-glutamine and Earle’s balanced salt solution adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 10% fetal bovine serum. Immunoprecipitations and Western Blot Analysis. Cells were washed twice with ice-cold phosphate-buffered saline and then lysed in ice-cold Triton lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 0.5 mmol/L ethylenediaminetetraacetic acid, 10% glycerol, 1% Triton X-100, 100 mmol/L NaF, 2 mmol/L Na3VO4, 10 mmol/L sodium pyrophosphate, 10 mmol/L phenylmethylsulfonylfluoride, 500 mol/L 4-(2-aminoethyl) benzenesulfonylfluoride, 150 nmol/L aprotinin, 1 mol/L E-64, and 1 mol/L leupeptin). The lysates were centrifuged at 14,000g for 10 minutes at 4°C, and the supernatants were used for further analysis. Protein concentrations were determined by a colorimetric assay19 by using bovine albumin as a protein standard. Cell lysates containing 5 g of protein were electrophoresed in 12% denaturing polyacrylamide gels (sodium dodecyl sulfate– polyacrylamide gel electrophoresis). For immunoprecipitations, equal amounts of protein (250-500 g) from each sample were precipitated by using 4 g/mL of antibody and captured by using protein A agarose beads. Immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and visualized by Western blotting. Membranes were blocked with 3% bovine serum albumin and probed with antibodies (PY99: 1:2,000; anti–IGF-1R -subunit: 1:2,000; anti-EGFR: 1:2,000, anti–IRS-1, anti–IRS-2, anti-Akt, anti-MAP kinase, and anti-active MAP kinase (E10): 1:1,000; and anti-Src: 1:1,000) followed by secondary horseradish peroxidase– conjugated goat anti-rabbit IgG or horse anti-mouse IgG antibodies. The membranes were visualized by enhanced chemilumines-
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cence and were quantified by National Institutes of Health (Bethesda, MD) image software. Confocal Immunofluorescence Microscopy. Cells were cultured on glass cover slips for 4 hours. The cover slips were rinsed briefly in phosphate-buffered saline (PBS) and then fixed in 4% phosphate-buffered formaldehyde solution for 20 minutes, washed extensively with PBS, and then incubated for 30 minutes in PBS supplemented with 10% goat serum at room temperature. Slips were then treated with anti-IGF-binding protein-3 (IGFBP-3) antibodies (Diagnostic System Laboratories, Webster, TX; 1:1,000 in 10% goat serum/PBS) for 45 minutes at 37°C. Slips were then washed 3 times in PBS (5 minutes each) and incubated with Alexa Fluor (Molecular Probe, Eugene, OR; 1:200 in 10% goat serum/PBS) for 45 minutes at 37°C. The slips were then washed in PBS and mounted on slides by using SlowFade (Molecular Probes). IGFBP-3 staining was visualized by using a BioRad (Hercules, CA) MRC1024/2P laser scanning confocal microscope focused on cell membrane, excitation 488 nm, emission 528 to 550 nm. All data are representative of one of at least 3 experiments.
Results EGF Induces IGF-IR Tyrosine Phosphorylation. Rat hepatocytes express IGF-IR,9-12 but the ability of IGF-I to initiate intracellular signaling is uncertain. The tyrosine phosphorylation status of the EGFR and the IGF-IR in suspensions of freshly isolated rat hepatocytes was investigated by immunoprecipitation and Western blot analysis. EGF stimulated EGFR tyrosine phosphorylation (seen as a doublet), which was inhibited by the EGFR-specific tyrphostin AG1478 (Fig. 1A).20 It has been reported that IGF-I can induce EGFR activation in COS-7 cells.21 However, this did not occur in rat hepatocytes. IGF-I did not induce IGF-IR tyrosine autophosphorylation (Fig. 1A). Neither a higher dose of IGF-I (200 ng/mL) nor prolonged incubation with the ligand (1 hour) were effective in activating the IGF-IR (data not shown). It has been reported that the IGF-IR can be transactivated by intracellular pathways.22-24 The ability of EGF to transactivate the IGF-IR was tested in hepatocytes. In contrast with the inactivity of IGF-I, EGF induced the tyrosine phosphorylation of the IGF-IR (Fig. 1A). IGF-IR also was identified in phosphotyrosine immunoprecipitates after EGF treatment (not shown). IGF-I did not enhance EGF-induced phosphorylation of the IGF-IR (not shown).
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IGF-IR tyrosine phosphorylation in response to EGF was inhibited by AG1478. I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR,25 also completely inhibited EGF-mediated transactivation of the IGF-IR, and had no effect on EGFR phosphorylation. The expression levels of the EGFR and IGF-IR were unchanged under any treatment condition. Transactivation of the IGF-IR by EGF was not observed in several other cell types including balb/c3T3 fibroblasts, cultured rat cerebellar granule neurons, and NG-108 neuroblastoma ⫻ glioma hybrid cells (data not shown). The intracellular  subunits of the IGF-IR and insulin receptor (IR) can exist as hybrids.26 We considered the possibility that the tyrosine-phosphorylated protein band corresponding to the IGF-IR included IR  subunits, which also might be transactivated by EGF. However, IR was not identified by Western immunoblotting of IGF-IR immunoprecipitates (data not shown). Moreover, EGF did not induce IR phosphorylation as determined by phosphotyrosine immunoblotting of IR immunoprecipitates (Fig. 1B). IGF-I had no effect on IR autophosphorylation. The time-courses of EGF-induced phosphorylations of EGFR and IGF-IR are shown in Fig. 2. Both receptors were phosphorylated maximally within 2 to 3 minutes, and remained at this level for 15 minutes. Phosphorylation of both receptors declined after 15 minutes, and approached near-basal levels by 1 hour. IGFBP-3 Inhibits IGF-I Activity. The inactivity of IGF-I might be due to an intracellular pool of the IGF-IR, inaccessible to IGF-I. However, it has been reported elsewhere9-12 that IGF-I does indeed bind weakly to isolated rat hepatocytes and to intact liver. By using fluorescence confocal microscopy, we observed surface staining with anti–IGF-IR antibody, which was not influenced by EGF (data not shown). We considered the possibility that inhibitory IGFbinding proteins (IGFBP) might interfere with the association of IGF-I with IGF-IR. IGFBP-3 in particular has been reported to inhibit IGF-IR activation.27,28 To explore this possibility, we treated hepatocytes with truncated des(1-3)-IGF-I, which has a 25- to 50-fold lower affinity for IGFBP-3 than IGF-I.29,30 As shown in Fig. 3, des(1-3)-IGF-I induced tyrosine phosphorylation of the IGF-IR, which was inhibited by I-Ome-AG538. Des(13)-IGF-I had no effect on EGFR phosphorylation (not shown). Confocal immunofluorescence microscopy showed surface association of the IGFBP-3 on hepatocytes (Fig. 4). IGF-IR Phosphorylation Is Dependent on Src Activation. Peterson et al.22,23 showed that activated Src kinase phosphorylates IGF-IR at its principal autophos-
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Fig. 1. EGF transactivates the IGF-IR. Freshly isolated rat hepatocytes were treated with either IGF-I, EGF, or insulin (20 ng/mL each). Where indicated, cells were treated with AG1478 (20 nmol/ L) or I-Ome-AG538 (20 nmol/L) for 10 minutes before the addition of EGF. Lysates were obtained after 10 minutes of ligand treatment. EGF-R and (A) IGF-IR or (B) IR were immunoprecipitated, and the extent of tyrosine phosphorylation was determined by Western blotting by using pY-99 antiphosphotyrosine antibodies, as described in the Materials and Methods section. Content of EGFR, IGF-IR, or IR within their respective immunoprecipitates was quantitated by using respective antibodies.
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phorylation sites, Y1131, Y1135, and Y1136. To investigate whether Src kinase transactivates the IGF-IR in rat hepatocytes, we used phenylpyrazole Src inhibitors PP-1 and PP-2. Fig. 5 shows that EGF-mediated phosphorylation of the IGF-IR was inhibited completely by both compounds.31 PP-3, an inactive phenylpyrazole compound,32 had no effect on IGF-IR transactivation. Neither PP-1 nor PP-2 affected EGFR phosphorylation or its expression. We next studied the effect of EGF on the dynamics of Src activation. Src tyrosine phosphorylation was measured by Western blotting by using Src pY215 antibody, which recognizes phosphorylated Y215 (Fig. 6). Total Src was quantified by using Src-2, which was raised against the carboxy terminus of c-Src p60, and reacts with c-Src and several Src family members. EGF, but not IGF-I, induced Src tyrosine phosphorylation. AG1478 prevented EGF-mediated activation of Src. PP-1 did not inhibit Src phosphorylation. Src, both total and tyrosinephosphorylated, was detected in immunoprecipitates of both IGF-IR and EGFR after EGF treatment (Fig. 7). PP-1 and AG1478 prevented Src association with the EGFR and IGF-IR. Taken together, the data show that EGF induces Src activation, which results in Src association with the IGF-IR. The use of Src inhibitors indicates a requirement for Src in IGF-IR activation. EGFR and IGF-IR tyrosine phosphorylation was investigated in human hepatocarcinoma-derived HepG2 cells (Fig. 8). In contrast with rat hepatocytes, EGF only minimally induced tyrosine autophosphorylation of the IGF-IR. Although EGF induced the phosphorylation of the EGFR in these cells (lower panel), we did not detect phospho-Src (pY215) in lysates from these cells, despite the presence of Src (c-Src p60; not shown). Role of IGF-IR in Intracellular Signaling. The role of IGF-IR in intracellular signal generation was investigated by using the IGF-IR inhibitor AG538. Fig. 9 shows the phosphorylations of IRS-1, IRS-2, Akt, and MAP kinase in response to EGF. IRS-1 and IRS-2 phosphorylations were inhibited completely by AG538, whereas the phosphorylation of Akt and MAPK was reduced by 42% ⫾ 8% and 38% ⫾ 9%, respectively (means ⫾ SD from 3 experiments). PP-1 inhibited the phosphorylation of these mediators to a similar extent as AG538. IGF-I had no effect on the phosphorylation of these mediators.
Discussion EGF-Mediated Transactivation of the IGF-IR, IRS-1, and IRS-2. EGF is a prototypic hepatic mitogen, most likely playing a role in the early phases of liver regeneration.7 The activated EGFR binds a series of signal-
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Fig. 2. Time-course of EGFR and IGF-IR phosphorylation. Hepatocytes were treated with 20 ng/mL EGF for the indicated times. IGF-IR (upper panel) and EGFR (lower panel) tyrosine phosphorylation and total content were measured by Western blotting.
ing molecules that bear Src-homology (SH-2 and SH-3) domains, including phosphatidylinositol 3-kinase, Grb2, phospholipase C-␥, and Gab2. The binding of Grb2SOS in particular leads to the activation of MAPK, via membrane-associated Ras. A role for IRS-1 in liver regeneration was suggested initially by Sasaki et al.33 IRS-1 expression was enhanced within 30 minutes of two-thirds partial hepatectomy. IRS-1 tyrosine phosphorylation also occurred with peaks identified at 1 and 12 hours after partial hepatectomy. It was reported recently that EGF also induces the phosphorylation of IRS-1 in cultured rat hepatocytes, although a specific mechanism was not identified.34
Fig. 3. Des(1-3)-IGF-I induces IGF-IR phosphorylation. Hepatocytes were treated with either IGF-I (20 ng/mL) or des(1-3)IGF-I (20 ng/mL). Lysates were obtained after 10 minutes, and tyrosine phosphate content within the IGF-IR was determined by Western blotting. Total content of IGF-IR within immunoprecipitates is shown in the lower panel.
Fig. 4. Surface association of IGFBP-3 on hepatocytes. Confocal immunofluorescence microscopy was used to detect IGFBP-3 in hepatocytes, as described in the Materials and Methods section. (A) Alexa fluorescein only; (B) anti–IGFBP-3 antibody.
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Fig. 5. IGF-IR phosphorylation is Src dependent. Hepatocytes were treated for 10 minutes with EGF (20 ng/mL) for 10 minutes in the presence or absence of PP-1, PP-2, or PP-3 (1 mol/L each). IGF-IR and EGFR were immunoprecipitated and probed for tyrosine phosphate and total content by Western blotting.
We now provide a mechanism for EGF-induced phosphorylation of IRS-1 and associated downstream signaling. EGF, in contrast with IGF-I, rapidly induced tyrosine phosphorylation of IGF-IR (Fig. 1). The transFig. 7. EGF induces the association of Src with IGF-IR and EGFR. Hepatocytes were treated with EGF (20 ng/mL) for 10 minutes in the presence or absence of either PP-1 (1 mol/L) or AG1478 (20 nmol/L). The associations of phospho-Src and total Src with the EGFR and IGF-IR were visualized in their respective immunoprecipitates by Western blotting.
Fig. 6. EGF induces Src phosphorylation. Hepatocytes were treated with either IGF-I (20 ng/mL) or EGF (20 ng/mL) for 10 minutes in the presence or absence of either PP-1 (1 mol/L) or AG1478 (20 nmol/L). Control incubations received no treatments. Cell lysates were probed for phospho-Src (Src [pY215]) (upper panel) or total content (Src-2) (lower panel) by Western blotting.
activation of IGF-IR was inhibited by the specific IGF-IR inhibitor AG538, but had no effect on EGFR phosphorylation. AG538 also variably inhibited EGF-induced phosphorylation of IRS-1, IRS-2, Akt, and p42/p44 MAP kinase (Fig. 9). We further show that Src kinase is a mediator of IGF-IR transactivation and IRS-1 and IRS-2 phosphorylation in response to EGF. An autocrine pathway for IGF-IR phosphorylation was considered. However, the inability of IGF-I to activate the IGF-IR in rat hepatocytes indicates that an autocrine pathway of IGF-I stimulation is unlikely to account for EGF transactivation of the IGF-IR. In the studies by Sasaki et al.,33 IR tyrosine autophosphorylation also occurred after partial hepatectomy, but
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significantly later (2 hours) than the early phase of IRS-1 phosphorylation (0.5 hours), raising the possibility of additional receptors involved in IRS-1 phosphorylation. The IGF-IR inhibitor AG538 also inhibits insulin receptor tyrosine kinase activity, albeit with lower efficiency.25 However, our data do not support a role for insulin receptor in EGF-induced signaling, at least during the early phases of EGF stimulation considered in this study. Importantly, the tyrosine phosphorylation status of the IR was unaffected by EGF (Fig. 1), and we did not detect IR  subunits within IGF-IR immunoprecipitates (data not shown). However, these data do not rule out a role for IR activity in later phases of hepatocyte signaling. Src Kinase Mediates IGF-IR Transactivation. Several lines of evidence link Src kinase activation to the transactivation of the IGF-IR in rat hepatocytes. First, EGF induced the phosphorylation of Src and its association with the IGF-IR (Figs. 6 and 7). Second, Src kinase inhibitors (PP-1, PP-2) prevented IGF-IR phosphorylation. Third, in HepG2 cells, EGF minimally activated the IGF-IR, and did not induce Src phosphorylation or its association with the IGF-IR (Fig. 8).
Fig. 9. IGF-IR– dependent phosphorylation of IRS-1, Akt, and MAP kinase. Hepatocytes were treated for 10 minutes with IGF-I or EGF (20 ng/mL each) in the presence or absence of either I-Ome-AG538 (20 nmol/L) or PP-1 (1 mol/L). Control incubations received no treatments. IRS-1 and IRS-2 were immunoprecipitated and immunoblotted for phosphotyrosine content. Lysates were probed for phospho-Akt and phosphop42/p44 MAPK. Total content of the respective proteins was unchanged by any treatment (not shown).
Fig. 8. EGF does not transactivate the IGF-IR in HepG2 cells. HepG2 cells were treated with EGF (20 ng/mL) or IGF-I (20 ng/mL). After 10 minutes, lysates were obtained, and the IGF-IR (upper panels) and EGFR (lower panels) were immunoprecipitated and probed for phosphotyrosine content and total content by Western blotting.
EGF induced the phosphorylation of Src Y215, which is found within the Src homology 2 binding pocket. It is postulated that Y215 phosphorylation disrupts Src homology 2 interaction with the C-terminal regulatory phosphotyrosine (Y527), which can lead to Src translocation.35 Interestingly, in hepatocytes, EGF did not induce phosphorylation of Src Y418 within the kinase domain (data not shown). This finding might suggest that Src tyrosine kinase activity is not strictly necessary for IGF-IR transactivation. Indeed, the kinase activity of Src is not essential for several of its functional properties, and translocated Src may serve as an adaptor protein, independent of its kinase function.36 However, in contrast to this interpretation, the inhibitory action of PP-1 on IGF-IR transactivation and Src translocation does suggest a role for Src tyrosine kinase in these activities. As for Src regulation in diverse cell types,35 resolution of these complex-
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ities in hepatocytes awaits a full description of Src tyrosine phosphorylation at multiple regulatory subunits. Such analysis also must take into consideration the role of protein tyrosine phosphatases, which affect the status of both Src and IGF-IR tyrosine phosphorylation. The existence of an intracellular transactivation pathway for IGF-IR is not without precedent. Murine fibroblasts with targeted knockout of the IGF-IR (R⫺ cells) are not responsive to the transforming properties of an overexpressed EGFR.24,37,38 EGFR-dependent transformation of R⫺ cells is restored on overexpression of a functional IGF-IR, and is associated with IGF-IR phosphorylation in an EGF-dependent manner.24 Transactivation of the IGF-IR appears to be required for transformation by several other transforming agents, including Src.22,38 Rao et al.39 showed that thrombin induces IGF-IR phosphorylation in rat aortic smooth muscle cells, which correlates with Src activation. IGF-IR–Independent Pathway of IRS-1 Phosphorylation. In contrast with the current study, Fujioka and Ui34 did not identify IGF-IR phosphorylation in response to EGF in rat hepatocytes, suggesting an additional IGFIR–independent mechanism of IRS-1 and IRS-2 activation by EGF. Rat strain differences aside, induction of IGF-IR phosphorylation by EGF was assessed after only 30 seconds in their study,34 a time at which IGF-IR phosphorylation was increased minimally in our study. It is curious that the IGF-IR in their hepatocyte cultures was quite sensitive to IGF-I, as were HepG2 cells in the current study. Assuming no contamination from nonparenchymal cells, these findings indicate that multiple factors discussed earlier might regulate the sensitivity of the IGF-IR to IGF-I. These differences notwithstanding, the current data do not rule out an IGF-IR–independent component of IRS-1 and IRS-2 phosphorylation. For example, Src kinase may directly target IRS-1 and IRS-2. Indeed, Src family member p59fyn has been reported to associate with IRS-1 via pleckstrin homology domain binding.40 We identified phospho-Src within IRS-1 immunoprecipitates from EGF-treated cells (not shown). However, given the numerous proteins that associate with IRS-1, we cannot conclude that Src directly associates with IRS-1, or for that matter, the IGF-IR. Hepatocyte Insensitivity to IGF-I. IGF-I has been reported to exert weak metabolic and mitogenic effects on primary cultured hepatocytes.13-16 It has been argued, however, that these biologic effects are mediated by IGF-I binding to insulin receptors.14,16,17 Indeed, in the current study, a 10-fold higher concentration of IGF-I weakly induced tyrosine autophosphorylation of the insulin re-
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ceptor (not shown). There also is a possibility that mitogenic effects of IGF-I in some studies reflects the IGF-I response of nonparenchymal hepatic cells such as endothelial cells41 and hepatic stellate cells.42,43 In the current study, hepatocytes were washed extensively during their preparation, and were free of nonparenchymal cells. The nonresponsiveness of our hepatocyte cultures to IGF-I in terms of IGF-IR phosphorylation further rules out the presence of nonparenchymal cells that might express IGFI–sensitive IGF-IR. From a functional perspective, hepatocyte insensitivity to IGF-I is understandable given the need to tightly control the quiescent state of the undamaged liver, particularly because the liver is the major source of serum IGF-I. Enhanced expression of the IGF-IR may be associated with malignant transformation. In a model of rat hepatocarcinogenesis,12 hepatocellular carcinoma showed an increase in IGF-IR expression. IGF-IR expression is 12-fold increased in HepG2 hepatocellular carcinoma cells compared with normal hepatocytes.9 Several cell culture model systems have indicated that mitogenesis requires IGF-I expression beyond a threshold level.1 The low level of hepatocyte IGF-IR expression9,10 may account, in part, for the inability of IGF-I to stimulate hepatocytes. However, this is not necessarily due to insensitivity of the IGF-IR to IGF-I. For example, serumstarved 3T3 cells do not proliferate in response to IGF-I, yet exhibit IGF-I– dependent IGF-IR phosphorylation.44 Rather, the current data strongly suggest that IGF-IR insensitivity to IGF-I is due to the prevention of IGF-I binding to the IGF-IR by IGFBPs.27 In particular, IGFBP-3 inhibits the proliferation of several cell types.11,28,45 Our current findings of IGF-IR activation by des(1-3)-IGF-I, and surface coating by IGFBP-3, support an inhibitory role for this class of IGFBP in hepatocytes. IGFBP-3 is synthesized by hepatic endothelial cells and Kupffer cells.46,47 Thus, it is likely that IGFBP-3 associates with hepatocytes, as evidenced by the current study. In summary, our findings provide insight into a novel pathway of IGF-IR phosphorylation in hepatocytes. The transactivation of the IGF-IR by EGF and Src provides a mechanism by which the IGF-IR could participate in intracellular signaling leading to mitogenesis. The survival properties of the IGF-IR also may play an important function during liver regeneration. In particular, the regenerating liver is stimulated by several cytokines with pleiotropic activities, notably tumor necrosis factor ␣. Activation of intracellular signaling pathways by the IGF-IR and other survival factors might diminish the apoptotic influences exerted by these cytokines.
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References 1. Rubin R, Baserga R. Insulin-like growth factor-I receptor. Its role in cell proliferation, apoptosis, and tumorigenicity. Lab Invest 1995;73:311-331. 2. LeRoith D, Werner H, Beitner-Johnson D, Roberts CT Jr. Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 1995;16:143-163. 3. Baserga R, Hongo A, Rubini M, Prisco M, Valentinis B. The IGF-I receptor in cell growth, transformation and apoptosis. Biochim Biophys Acta 1997;1332:F105-F126. 4. LeRoith D. Insulin-like growth factor I receptor signaling— overlapping or redundant pathways? Endocrinology 2000;141:1287-1288. 5. Brunet A, Datta SR, Greenberg ME. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol 2001;11:297-305. 6. Galetic I, Andjelkovic M, Meier R, Brodbeck D, Park J, Hemmings BA. Mechanism of protein kinase B activation by insulin/insulin-like growth factor-1 revealed by specific inhibitors of phosphoinositide 3-kinase— significance for diabetes and cancer. Pharmacol Ther 1999;82:409-425. 7. Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997; 276:60-66. 8. Le Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis: 2001. Endocr Rev 2001;22:53-74. 9. Caro JF, Poulos J, Ittoop O, Pories WJ, Flickinger EG, Sinha MK. Insulinlike growth factor I binding in hepatocytes from human liver, human hepatoma, and normal, regenerating, and fetal rat liver. J Clin Invest 1988; 81:976-981. 10. Santos A, Yusta B, Fernandez-Moreno MD, Blazquez E. Expression of insulin-like growth factor-I (IGF-I) receptor gene in rat brain and liver during development and in regenerating adult rat liver. Mol Cell Endocrinol 1994;101:85-93. 11. Barreca A, Voci A, Minuto F, de Marchis M, Cecchelli E, Fugassa E, Giordano G, et al. Effect of epidermal growth factor on insulin-like growth factor-I (IGF- I) and IGF-binding protein synthesis by adult rat hepatocytes. Mol Cell Endocrinol 1992;84:119-126. 12. Scharf JG, Ramadori G, Dombrowski F. Analysis of the IGF axis in preneoplastic hepatic foci and hepatocellular neoplasms developing after lownumber pancreatic islet transplantation into the livers of streptozotocin diabetic rats. Lab Invest 2000;80:1399-1411. 13. Grunnet N, Peng X, Tygstrup N. Growth factors and gene expression in cultured rat hepatocytes. J Hepatol 1999;31:117-122. 14. Hartmann H, Schmitz F, Christ B, Jungermann K, Creutzfeldt W. Metabolic actions of insulin-like growth factor-I in cultured hepatocytes from adult rats. HEPATOLOGY 1990;12:1139-1143. 15. Kimura M, Ogihara M. Effects of insulin-like growth factor I and II on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Eur J Pharmacol 1998;354:271-281. 16. Liu YY, Rajkumar K, Murphy LJ. Hepatic regeneration in insulin-like growth factor binding protein-1 transgenic mice. J Hepatol 1999;30:674680. 17. Parkes JL, Cardell RR, Grieninger G. Insulin-like growth factors (IGF I and IGF II) mimic the effect of insulin on plasma protein synthesis and glycogen deposition in cultured hepatocytes. Biochem Biophys Res Commun 1986;134:427-435. 18. Hoek JB, Thomas AP, Rubin R, Rubin E. Ethanol-induced mobilization of calcium by activation of phosphoinositide-specific phospholipase C in intact hepatocytes. J Biol Chem 1987;262:682-691. 19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. 20. Levitzki A, Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 1995;267:1782-1788. 21. Roudabush FL, Pierce KL, Maudsley S, Khan KD, Luttrell LM. Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells. J Biol Chem 2000;275: 22583-22589.
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22. Peterson JE, Kulik G, Jelinek T, Reuter CW, Shannon JA, Weber MJ. Src phosphorylates the insulin-like growth factor type I receptor on the autophosphorylation sites. Requirement for transformation by src. J Biol Chem 1996;271:31562-31571. 23. Peterson JE, Jelinek T, Kaleko M, Siddle K, Weber MJ. c phosphorylation and activation of the IGF-I receptor in src- transformed cells. J Biol Chem 1994;269:27315-27321. 24. Burgaud JL, Baserga R. Intracellular transactivation of the insulin-like growth factor I receptor by an epidermal growth factor receptor. Exp Cell Res 1996;223:412-419. 25. Blum G, Gazit A, Levitzki A. Substrate competitive inhibitors of IGF-1 receptor kinase. Biochemistry 2000;39:15705-15712. 26. Treadway JL, Morrison BD, Soos MA, Siddle K, Olefsky J, Ullrich A, McClain DA, et al. Transdominant inhibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors. Proc Natl Acad Sci U S A 1991;88:214-218. 27. Grimberg A, Cohen P. Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis. J Cell Physiol 2000;183: 1-9. 28. Karas M, Danilenko M, Fishman D, LeRoith D, Levy J, Sharoni Y. Membrane-associated insulin-like growth factor-binding protein-3 inhibits insulin-like growth factor-I-induced insulin-like growth factor-I receptor signaling in ishikawa endometrial cancer cells. J Biol Chem 1997;272: 16514-16520. 29. Lord AP, Bastian SE, Read LC, Walton PE, Ballard FJ. Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins. J Endocrinol 1994;140:475-482. 30. Heding A, Gill R, Ogawa Y, De Meyts P, Shymko RM. Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3. J Biol Chem 1996;271:13948-13952. 31. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, et al. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 1996;271:695-701. 32. Traxler P, Bold G, Frei J, Lang M, Lydon N, Mett H, Buchdunger E, et al. Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines. J Med Chem 1997;40:3601-3616. 33. Sasaki Y, Zhang XF, Nishiyama M, Avruch J, Wands JR. Expression and phosphorylation of insulin receptor substrate 1 during rat liver regeneration. J Biol Chem 1993;268:3805-3808. 34. Fujioka T, Ui M. Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3-kinase in rat hepatocyte primary culture. Eur J Biochem 2001;268:25-34. 35. Schwartzberg PL. The many faces of Src: multiple functions of a prototypical tyrosine kinase. Oncogene 1998;17:1463-1468. 36. Stover DR, Furet P, Lydon NB. Modulation of the SH2 binding specificity and kinase activity of Src by tyrosine phosphorylation within its SH2 domain. J Biol Chem 1996;271:12481-12487. 37. Coppola D, Ferber A, Miura M, Sell C, D’Ambrosio C, Rubin R, Baserga R. A functional insulin-like growth factor I receptor is required for the mitogenic and transforming activities of the epidermal growth factor receptor. Mol Cell Biol 1994;14:4588-4595. 38. Baserga R, Resnicoff M, Dews M. The IGF-I receptor and cancer. Endocrine 1997;7:99-102. 39. Rao GN, Delafontaine P, Runge MS. Thrombin stimulates phosphorylation of insulin-like growth factor-1 receptor, insulin receptor substrate-1, and phospholipase C-gamma 1 in rat aortic smooth muscle cells. J Biol Chem 1995;270:27871-27875. 40. Sun XJ, Pons S, Asano T, Myers MG Jr, Glasheen E, White MF. The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation. J Biol Chem 1996;271:10583-10587. 41. Liu W, Liu Y, Lowe JW Jr. The role of phosphatidylinositol 3-kinase and the mitogen-activated protein kinases in insulin-like growth factor-I-me-
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diated effects in vascular endothelial cells. Endocrinology 2001;142:17101719. 42. Scharf JG, Knittel T, Dombrowski F, Muller L, Saile B, Braulke T, Hartman H, et al. Characterization of the IGF axis components in isolated rat hepatic stellate cells. HEPATOLOGY 1998;27:1275-1284. 43. Gressner AM, Lahme B, Brenzel A. Molecular dissection of the mitogenic effect of hepatocytes on cultured hepatic stellate cells. HEPATOLOGY 1995; 22:1507-1518. 44. Seiler AE, Ross BN, Green JS, Rubin R. Differential effects of ethanol on insulin-like growth factor-I receptor signaling. Alcohol Clin Exp Res 2000; 24:140-148.
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45. Li YM, Schacher DH, Liu Q, Arkins S, Rebeiz N, McCusker RH Jr, Dantzer R, et al. Regulation of myeloid growth and differentiation by the insulin-like growth factor I receptor. Endocrinology 1997;138:362-368. 46. Chin E, Zhou J, Dai J, Baxter RC, Bondy CA. Cellular localization and regulation of gene expression for components of the insulin-like growth factor ternary binding protein complex. Endocrinology 1994;134:24982504. 47. Arany E, Afford S, Strain AJ, Winwood PJ, Arthur MJ, Hill DJ. Differential cellular synthesis of insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 within human liver. J Clin Endocrinol Metab 1994; 79:1871-1876.
I
nsulin-like growth factor I (IGF-I) is a critical regulator of cell proliferation and survival.1,2 For many cell types, IGF-I induces mitogenesis, and also is a direct inhibitor of apoptosis. IGF-I signaling is initiated by high-affinity binding to the IGF-I receptor (IGF-IR). The IGF-IR exists as an ␣2-2 heterodimer. The extracellular ligand-binding domain is constituted on the ␣ chain, and the intracellular  domain contains a classic tyrosine kinase that is over 80% homologous to the insu-
Abbreviations: IGF-I, insulin-like growth factor I; IFT-IR, insulin-like growth factor I receptor; IRS, insulin-related substrates; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Ig, immunoglobulin; PBS, phosphate-buffered saline; IGFBP-3, insulin-like growth factor binding protein-3; IR, insulin receptor. From the Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA. Received June 20, 2002; accepted September 15, 2002. Supported by National Institutes of Health grants AA09976, AA07186, and AA08714. Address reprint requests to: Raphael Rubin, M.D., Department of Pathology, Anatomy and Cell Biology, 1020 Locust St., Room 226 Alumni Hall, Philadelphia, PA 19107. E-mail: [email protected]; fax: 215-955-8703. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3606-0027$35.00/0 doi:10.1053/jhep.2002.37138
lin receptor tyrosine kinase. On ligand binding, the receptor undergoes autophosphorylation within the tyrosine kinase domain. Other key tyrosines that are important for downstream signaling are located in the juxtamembrane region and within the carboxyterminal domain. The roles of the respective tyrosines in cell proliferation, survival, and transformation have been dissected by means of sitedirected mutagenesis, and have been reviewed elsewhere.3 Intracellular signaling by the IGF-IR is exceedingly similar to that of the insulin receptor.2,4 After IGF-I binding, the IGF-IR undergoes rapid tyrosine autophosphorylation, and then phosphorylates key signaling molecules including a family of insulin-related substrates (IRS) and Shc. Tyrosine-phosphorylated IRS-1, IRS-2, and Shc bind several molecules that bear Src homology 2 adaptor sites such as Grb2, which leads to activation of p21ras and the mitogen-activated protein kinases (MAPK). The antiapoptotic properties of the IGF-IR principally are mediated by IRS-1 binding to the p85 subunit of phosphatidylinositol 3-kinase, and the subsequent activation of Akt/PKB.5,6 Liver regeneration is a process that is controlled tightly by an array of cytokines and growth factors.7 Factors that 1509
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have been identified that prime hepatocytes for mitogenesis include tumor necrosis factor ␣ and interleukin-6. Hepatic mitogens that induce G1-S phase transition include hepatocyte growth factor, transforming growth factor , and epidermal growth factor (EGF). The mitogenic action of these growth factors is enhanced by comitogens including insulin, glucagon, norepinephrine, thyroid hormone, parathormone, prolactin, and estrogen. In contrast with most cell types, a definitive role for IGF-I in hepatocyte growth has not been delineated. The liver is the major source of circulating IGF-I8 but there is negligible IGF-I binding to hepatocytes.9-12 IGF-I has been reported to induce modest metabolic effects and only slight enhancement of DNA synthesis in cultured hepatocytes,13-15 which likely is mediated by IGF-I binding to insulin receptors.14,16,17 IGF-I binding to rat liver plasma membranes and isolated hepatocytes increases after several days of liver regeneration.9,10 In this study, we have assessed the potential signaling role of the IGF-IR in rat hepatocytes. We confirm the inactivity of IGF-I toward the IGF-IR in freshly isolated rat hepatocytes and identify an inhibitory role for surfaceassociated IGF-I binding protein-3. In contrast, EGF induces the rapid tyrosine phosphorylation of the IGF-IR, a process that is dependent on Src kinase activation. We further show a role for the transactivated IGF-IR in the phosphorylation of key downstream signaling mediators.
Materials and Methods Materials. Human recombinant IGF-I was purchased from Calbiochem (San Diego, CA). EGF was purchased from Sigma (St. Louis, MO). Des(1-3)-IGF-I was obtained from Gropep (Adelaide, Australia). Anti–IGFIR -subunit rabbit polyclonal antibodies and antiphosphotyrosine (PY99) mouse monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGF receptor (EGFR) antibodies and the EGFR antagonist AG1478 were obtained from Calbiochem. Anti–IRS-1 and anti–IRS-2 antibodies (rabbit, polyclonal immunoglobulin [Ig]G) were obtained from UBI (Lake Placid, NY). Anti-MAP kinase antibodies, anti-phosphotyrosine and anti-phosphothreonine MAP kinase (E10) antibodies, horseradish peroxidase– conjugated goat anti-rabbit IgG, horse anti-mouse IgG antibodies, and signal-enhanced chemiluminescence reagents were obtained from New England Biolabs (Beverly, MA). Anti– c-Src antibody (Src-2; sc-18) was purchased from Santa Cruz Biotechnology, and anti–phospho-Src antibodies (pY215) were purchased from Biosource (Camarillo, CA). Anti-Akt antibodies were from Cell Signaling (Beverly, MA). Pyrazolopyrimidines PP-1, PP-2, and
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PP-3 were purchased from Biomol (Plymouth Meeting, PA). Tyrphostin I-Ome-AG538 was a generous gift from Dr. A. Levitzki, Hebrew University, Jerusalem. All other chemicals and biochemicals were of the highest purity commercially available. Hepatocyte Preparation and Culture Conditions. Hepatocytes were isolated by collagenase perfusion as described previously.18 Cells were washed 4 times to remove nonparenchymal cells, which was verified by visual inspection. Freshly isolated hepatocytes were cultured for 2 hours in serum-free Williams medium E at 37°C, 5% CO2, before the addition of growth factors. HepG2 cells were purchased from ATCC (Manassas, VA), and were grown in minimum essential Eagle medium with 2 mmol/L L-glutamine and Earle’s balanced salt solution adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 10% fetal bovine serum. Immunoprecipitations and Western Blot Analysis. Cells were washed twice with ice-cold phosphate-buffered saline and then lysed in ice-cold Triton lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 0.5 mmol/L ethylenediaminetetraacetic acid, 10% glycerol, 1% Triton X-100, 100 mmol/L NaF, 2 mmol/L Na3VO4, 10 mmol/L sodium pyrophosphate, 10 mmol/L phenylmethylsulfonylfluoride, 500 mol/L 4-(2-aminoethyl) benzenesulfonylfluoride, 150 nmol/L aprotinin, 1 mol/L E-64, and 1 mol/L leupeptin). The lysates were centrifuged at 14,000g for 10 minutes at 4°C, and the supernatants were used for further analysis. Protein concentrations were determined by a colorimetric assay19 by using bovine albumin as a protein standard. Cell lysates containing 5 g of protein were electrophoresed in 12% denaturing polyacrylamide gels (sodium dodecyl sulfate– polyacrylamide gel electrophoresis). For immunoprecipitations, equal amounts of protein (250-500 g) from each sample were precipitated by using 4 g/mL of antibody and captured by using protein A agarose beads. Immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and visualized by Western blotting. Membranes were blocked with 3% bovine serum albumin and probed with antibodies (PY99: 1:2,000; anti–IGF-1R -subunit: 1:2,000; anti-EGFR: 1:2,000, anti–IRS-1, anti–IRS-2, anti-Akt, anti-MAP kinase, and anti-active MAP kinase (E10): 1:1,000; and anti-Src: 1:1,000) followed by secondary horseradish peroxidase– conjugated goat anti-rabbit IgG or horse anti-mouse IgG antibodies. The membranes were visualized by enhanced chemilumines-
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cence and were quantified by National Institutes of Health (Bethesda, MD) image software. Confocal Immunofluorescence Microscopy. Cells were cultured on glass cover slips for 4 hours. The cover slips were rinsed briefly in phosphate-buffered saline (PBS) and then fixed in 4% phosphate-buffered formaldehyde solution for 20 minutes, washed extensively with PBS, and then incubated for 30 minutes in PBS supplemented with 10% goat serum at room temperature. Slips were then treated with anti-IGF-binding protein-3 (IGFBP-3) antibodies (Diagnostic System Laboratories, Webster, TX; 1:1,000 in 10% goat serum/PBS) for 45 minutes at 37°C. Slips were then washed 3 times in PBS (5 minutes each) and incubated with Alexa Fluor (Molecular Probe, Eugene, OR; 1:200 in 10% goat serum/PBS) for 45 minutes at 37°C. The slips were then washed in PBS and mounted on slides by using SlowFade (Molecular Probes). IGFBP-3 staining was visualized by using a BioRad (Hercules, CA) MRC1024/2P laser scanning confocal microscope focused on cell membrane, excitation 488 nm, emission 528 to 550 nm. All data are representative of one of at least 3 experiments.
Results EGF Induces IGF-IR Tyrosine Phosphorylation. Rat hepatocytes express IGF-IR,9-12 but the ability of IGF-I to initiate intracellular signaling is uncertain. The tyrosine phosphorylation status of the EGFR and the IGF-IR in suspensions of freshly isolated rat hepatocytes was investigated by immunoprecipitation and Western blot analysis. EGF stimulated EGFR tyrosine phosphorylation (seen as a doublet), which was inhibited by the EGFR-specific tyrphostin AG1478 (Fig. 1A).20 It has been reported that IGF-I can induce EGFR activation in COS-7 cells.21 However, this did not occur in rat hepatocytes. IGF-I did not induce IGF-IR tyrosine autophosphorylation (Fig. 1A). Neither a higher dose of IGF-I (200 ng/mL) nor prolonged incubation with the ligand (1 hour) were effective in activating the IGF-IR (data not shown). It has been reported that the IGF-IR can be transactivated by intracellular pathways.22-24 The ability of EGF to transactivate the IGF-IR was tested in hepatocytes. In contrast with the inactivity of IGF-I, EGF induced the tyrosine phosphorylation of the IGF-IR (Fig. 1A). IGF-IR also was identified in phosphotyrosine immunoprecipitates after EGF treatment (not shown). IGF-I did not enhance EGF-induced phosphorylation of the IGF-IR (not shown).
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IGF-IR tyrosine phosphorylation in response to EGF was inhibited by AG1478. I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR,25 also completely inhibited EGF-mediated transactivation of the IGF-IR, and had no effect on EGFR phosphorylation. The expression levels of the EGFR and IGF-IR were unchanged under any treatment condition. Transactivation of the IGF-IR by EGF was not observed in several other cell types including balb/c3T3 fibroblasts, cultured rat cerebellar granule neurons, and NG-108 neuroblastoma ⫻ glioma hybrid cells (data not shown). The intracellular  subunits of the IGF-IR and insulin receptor (IR) can exist as hybrids.26 We considered the possibility that the tyrosine-phosphorylated protein band corresponding to the IGF-IR included IR  subunits, which also might be transactivated by EGF. However, IR was not identified by Western immunoblotting of IGF-IR immunoprecipitates (data not shown). Moreover, EGF did not induce IR phosphorylation as determined by phosphotyrosine immunoblotting of IR immunoprecipitates (Fig. 1B). IGF-I had no effect on IR autophosphorylation. The time-courses of EGF-induced phosphorylations of EGFR and IGF-IR are shown in Fig. 2. Both receptors were phosphorylated maximally within 2 to 3 minutes, and remained at this level for 15 minutes. Phosphorylation of both receptors declined after 15 minutes, and approached near-basal levels by 1 hour. IGFBP-3 Inhibits IGF-I Activity. The inactivity of IGF-I might be due to an intracellular pool of the IGF-IR, inaccessible to IGF-I. However, it has been reported elsewhere9-12 that IGF-I does indeed bind weakly to isolated rat hepatocytes and to intact liver. By using fluorescence confocal microscopy, we observed surface staining with anti–IGF-IR antibody, which was not influenced by EGF (data not shown). We considered the possibility that inhibitory IGFbinding proteins (IGFBP) might interfere with the association of IGF-I with IGF-IR. IGFBP-3 in particular has been reported to inhibit IGF-IR activation.27,28 To explore this possibility, we treated hepatocytes with truncated des(1-3)-IGF-I, which has a 25- to 50-fold lower affinity for IGFBP-3 than IGF-I.29,30 As shown in Fig. 3, des(1-3)-IGF-I induced tyrosine phosphorylation of the IGF-IR, which was inhibited by I-Ome-AG538. Des(13)-IGF-I had no effect on EGFR phosphorylation (not shown). Confocal immunofluorescence microscopy showed surface association of the IGFBP-3 on hepatocytes (Fig. 4). IGF-IR Phosphorylation Is Dependent on Src Activation. Peterson et al.22,23 showed that activated Src kinase phosphorylates IGF-IR at its principal autophos-
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Fig. 1. EGF transactivates the IGF-IR. Freshly isolated rat hepatocytes were treated with either IGF-I, EGF, or insulin (20 ng/mL each). Where indicated, cells were treated with AG1478 (20 nmol/ L) or I-Ome-AG538 (20 nmol/L) for 10 minutes before the addition of EGF. Lysates were obtained after 10 minutes of ligand treatment. EGF-R and (A) IGF-IR or (B) IR were immunoprecipitated, and the extent of tyrosine phosphorylation was determined by Western blotting by using pY-99 antiphosphotyrosine antibodies, as described in the Materials and Methods section. Content of EGFR, IGF-IR, or IR within their respective immunoprecipitates was quantitated by using respective antibodies.
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phorylation sites, Y1131, Y1135, and Y1136. To investigate whether Src kinase transactivates the IGF-IR in rat hepatocytes, we used phenylpyrazole Src inhibitors PP-1 and PP-2. Fig. 5 shows that EGF-mediated phosphorylation of the IGF-IR was inhibited completely by both compounds.31 PP-3, an inactive phenylpyrazole compound,32 had no effect on IGF-IR transactivation. Neither PP-1 nor PP-2 affected EGFR phosphorylation or its expression. We next studied the effect of EGF on the dynamics of Src activation. Src tyrosine phosphorylation was measured by Western blotting by using Src pY215 antibody, which recognizes phosphorylated Y215 (Fig. 6). Total Src was quantified by using Src-2, which was raised against the carboxy terminus of c-Src p60, and reacts with c-Src and several Src family members. EGF, but not IGF-I, induced Src tyrosine phosphorylation. AG1478 prevented EGF-mediated activation of Src. PP-1 did not inhibit Src phosphorylation. Src, both total and tyrosinephosphorylated, was detected in immunoprecipitates of both IGF-IR and EGFR after EGF treatment (Fig. 7). PP-1 and AG1478 prevented Src association with the EGFR and IGF-IR. Taken together, the data show that EGF induces Src activation, which results in Src association with the IGF-IR. The use of Src inhibitors indicates a requirement for Src in IGF-IR activation. EGFR and IGF-IR tyrosine phosphorylation was investigated in human hepatocarcinoma-derived HepG2 cells (Fig. 8). In contrast with rat hepatocytes, EGF only minimally induced tyrosine autophosphorylation of the IGF-IR. Although EGF induced the phosphorylation of the EGFR in these cells (lower panel), we did not detect phospho-Src (pY215) in lysates from these cells, despite the presence of Src (c-Src p60; not shown). Role of IGF-IR in Intracellular Signaling. The role of IGF-IR in intracellular signal generation was investigated by using the IGF-IR inhibitor AG538. Fig. 9 shows the phosphorylations of IRS-1, IRS-2, Akt, and MAP kinase in response to EGF. IRS-1 and IRS-2 phosphorylations were inhibited completely by AG538, whereas the phosphorylation of Akt and MAPK was reduced by 42% ⫾ 8% and 38% ⫾ 9%, respectively (means ⫾ SD from 3 experiments). PP-1 inhibited the phosphorylation of these mediators to a similar extent as AG538. IGF-I had no effect on the phosphorylation of these mediators.
Discussion EGF-Mediated Transactivation of the IGF-IR, IRS-1, and IRS-2. EGF is a prototypic hepatic mitogen, most likely playing a role in the early phases of liver regeneration.7 The activated EGFR binds a series of signal-
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Fig. 2. Time-course of EGFR and IGF-IR phosphorylation. Hepatocytes were treated with 20 ng/mL EGF for the indicated times. IGF-IR (upper panel) and EGFR (lower panel) tyrosine phosphorylation and total content were measured by Western blotting.
ing molecules that bear Src-homology (SH-2 and SH-3) domains, including phosphatidylinositol 3-kinase, Grb2, phospholipase C-␥, and Gab2. The binding of Grb2SOS in particular leads to the activation of MAPK, via membrane-associated Ras. A role for IRS-1 in liver regeneration was suggested initially by Sasaki et al.33 IRS-1 expression was enhanced within 30 minutes of two-thirds partial hepatectomy. IRS-1 tyrosine phosphorylation also occurred with peaks identified at 1 and 12 hours after partial hepatectomy. It was reported recently that EGF also induces the phosphorylation of IRS-1 in cultured rat hepatocytes, although a specific mechanism was not identified.34
Fig. 3. Des(1-3)-IGF-I induces IGF-IR phosphorylation. Hepatocytes were treated with either IGF-I (20 ng/mL) or des(1-3)IGF-I (20 ng/mL). Lysates were obtained after 10 minutes, and tyrosine phosphate content within the IGF-IR was determined by Western blotting. Total content of IGF-IR within immunoprecipitates is shown in the lower panel.
Fig. 4. Surface association of IGFBP-3 on hepatocytes. Confocal immunofluorescence microscopy was used to detect IGFBP-3 in hepatocytes, as described in the Materials and Methods section. (A) Alexa fluorescein only; (B) anti–IGFBP-3 antibody.
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Fig. 5. IGF-IR phosphorylation is Src dependent. Hepatocytes were treated for 10 minutes with EGF (20 ng/mL) for 10 minutes in the presence or absence of PP-1, PP-2, or PP-3 (1 mol/L each). IGF-IR and EGFR were immunoprecipitated and probed for tyrosine phosphate and total content by Western blotting.
We now provide a mechanism for EGF-induced phosphorylation of IRS-1 and associated downstream signaling. EGF, in contrast with IGF-I, rapidly induced tyrosine phosphorylation of IGF-IR (Fig. 1). The transFig. 7. EGF induces the association of Src with IGF-IR and EGFR. Hepatocytes were treated with EGF (20 ng/mL) for 10 minutes in the presence or absence of either PP-1 (1 mol/L) or AG1478 (20 nmol/L). The associations of phospho-Src and total Src with the EGFR and IGF-IR were visualized in their respective immunoprecipitates by Western blotting.
Fig. 6. EGF induces Src phosphorylation. Hepatocytes were treated with either IGF-I (20 ng/mL) or EGF (20 ng/mL) for 10 minutes in the presence or absence of either PP-1 (1 mol/L) or AG1478 (20 nmol/L). Control incubations received no treatments. Cell lysates were probed for phospho-Src (Src [pY215]) (upper panel) or total content (Src-2) (lower panel) by Western blotting.
activation of IGF-IR was inhibited by the specific IGF-IR inhibitor AG538, but had no effect on EGFR phosphorylation. AG538 also variably inhibited EGF-induced phosphorylation of IRS-1, IRS-2, Akt, and p42/p44 MAP kinase (Fig. 9). We further show that Src kinase is a mediator of IGF-IR transactivation and IRS-1 and IRS-2 phosphorylation in response to EGF. An autocrine pathway for IGF-IR phosphorylation was considered. However, the inability of IGF-I to activate the IGF-IR in rat hepatocytes indicates that an autocrine pathway of IGF-I stimulation is unlikely to account for EGF transactivation of the IGF-IR. In the studies by Sasaki et al.,33 IR tyrosine autophosphorylation also occurred after partial hepatectomy, but
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significantly later (2 hours) than the early phase of IRS-1 phosphorylation (0.5 hours), raising the possibility of additional receptors involved in IRS-1 phosphorylation. The IGF-IR inhibitor AG538 also inhibits insulin receptor tyrosine kinase activity, albeit with lower efficiency.25 However, our data do not support a role for insulin receptor in EGF-induced signaling, at least during the early phases of EGF stimulation considered in this study. Importantly, the tyrosine phosphorylation status of the IR was unaffected by EGF (Fig. 1), and we did not detect IR  subunits within IGF-IR immunoprecipitates (data not shown). However, these data do not rule out a role for IR activity in later phases of hepatocyte signaling. Src Kinase Mediates IGF-IR Transactivation. Several lines of evidence link Src kinase activation to the transactivation of the IGF-IR in rat hepatocytes. First, EGF induced the phosphorylation of Src and its association with the IGF-IR (Figs. 6 and 7). Second, Src kinase inhibitors (PP-1, PP-2) prevented IGF-IR phosphorylation. Third, in HepG2 cells, EGF minimally activated the IGF-IR, and did not induce Src phosphorylation or its association with the IGF-IR (Fig. 8).
Fig. 9. IGF-IR– dependent phosphorylation of IRS-1, Akt, and MAP kinase. Hepatocytes were treated for 10 minutes with IGF-I or EGF (20 ng/mL each) in the presence or absence of either I-Ome-AG538 (20 nmol/L) or PP-1 (1 mol/L). Control incubations received no treatments. IRS-1 and IRS-2 were immunoprecipitated and immunoblotted for phosphotyrosine content. Lysates were probed for phospho-Akt and phosphop42/p44 MAPK. Total content of the respective proteins was unchanged by any treatment (not shown).
Fig. 8. EGF does not transactivate the IGF-IR in HepG2 cells. HepG2 cells were treated with EGF (20 ng/mL) or IGF-I (20 ng/mL). After 10 minutes, lysates were obtained, and the IGF-IR (upper panels) and EGFR (lower panels) were immunoprecipitated and probed for phosphotyrosine content and total content by Western blotting.
EGF induced the phosphorylation of Src Y215, which is found within the Src homology 2 binding pocket. It is postulated that Y215 phosphorylation disrupts Src homology 2 interaction with the C-terminal regulatory phosphotyrosine (Y527), which can lead to Src translocation.35 Interestingly, in hepatocytes, EGF did not induce phosphorylation of Src Y418 within the kinase domain (data not shown). This finding might suggest that Src tyrosine kinase activity is not strictly necessary for IGF-IR transactivation. Indeed, the kinase activity of Src is not essential for several of its functional properties, and translocated Src may serve as an adaptor protein, independent of its kinase function.36 However, in contrast to this interpretation, the inhibitory action of PP-1 on IGF-IR transactivation and Src translocation does suggest a role for Src tyrosine kinase in these activities. As for Src regulation in diverse cell types,35 resolution of these complex-
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ities in hepatocytes awaits a full description of Src tyrosine phosphorylation at multiple regulatory subunits. Such analysis also must take into consideration the role of protein tyrosine phosphatases, which affect the status of both Src and IGF-IR tyrosine phosphorylation. The existence of an intracellular transactivation pathway for IGF-IR is not without precedent. Murine fibroblasts with targeted knockout of the IGF-IR (R⫺ cells) are not responsive to the transforming properties of an overexpressed EGFR.24,37,38 EGFR-dependent transformation of R⫺ cells is restored on overexpression of a functional IGF-IR, and is associated with IGF-IR phosphorylation in an EGF-dependent manner.24 Transactivation of the IGF-IR appears to be required for transformation by several other transforming agents, including Src.22,38 Rao et al.39 showed that thrombin induces IGF-IR phosphorylation in rat aortic smooth muscle cells, which correlates with Src activation. IGF-IR–Independent Pathway of IRS-1 Phosphorylation. In contrast with the current study, Fujioka and Ui34 did not identify IGF-IR phosphorylation in response to EGF in rat hepatocytes, suggesting an additional IGFIR–independent mechanism of IRS-1 and IRS-2 activation by EGF. Rat strain differences aside, induction of IGF-IR phosphorylation by EGF was assessed after only 30 seconds in their study,34 a time at which IGF-IR phosphorylation was increased minimally in our study. It is curious that the IGF-IR in their hepatocyte cultures was quite sensitive to IGF-I, as were HepG2 cells in the current study. Assuming no contamination from nonparenchymal cells, these findings indicate that multiple factors discussed earlier might regulate the sensitivity of the IGF-IR to IGF-I. These differences notwithstanding, the current data do not rule out an IGF-IR–independent component of IRS-1 and IRS-2 phosphorylation. For example, Src kinase may directly target IRS-1 and IRS-2. Indeed, Src family member p59fyn has been reported to associate with IRS-1 via pleckstrin homology domain binding.40 We identified phospho-Src within IRS-1 immunoprecipitates from EGF-treated cells (not shown). However, given the numerous proteins that associate with IRS-1, we cannot conclude that Src directly associates with IRS-1, or for that matter, the IGF-IR. Hepatocyte Insensitivity to IGF-I. IGF-I has been reported to exert weak metabolic and mitogenic effects on primary cultured hepatocytes.13-16 It has been argued, however, that these biologic effects are mediated by IGF-I binding to insulin receptors.14,16,17 Indeed, in the current study, a 10-fold higher concentration of IGF-I weakly induced tyrosine autophosphorylation of the insulin re-
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ceptor (not shown). There also is a possibility that mitogenic effects of IGF-I in some studies reflects the IGF-I response of nonparenchymal hepatic cells such as endothelial cells41 and hepatic stellate cells.42,43 In the current study, hepatocytes were washed extensively during their preparation, and were free of nonparenchymal cells. The nonresponsiveness of our hepatocyte cultures to IGF-I in terms of IGF-IR phosphorylation further rules out the presence of nonparenchymal cells that might express IGFI–sensitive IGF-IR. From a functional perspective, hepatocyte insensitivity to IGF-I is understandable given the need to tightly control the quiescent state of the undamaged liver, particularly because the liver is the major source of serum IGF-I. Enhanced expression of the IGF-IR may be associated with malignant transformation. In a model of rat hepatocarcinogenesis,12 hepatocellular carcinoma showed an increase in IGF-IR expression. IGF-IR expression is 12-fold increased in HepG2 hepatocellular carcinoma cells compared with normal hepatocytes.9 Several cell culture model systems have indicated that mitogenesis requires IGF-I expression beyond a threshold level.1 The low level of hepatocyte IGF-IR expression9,10 may account, in part, for the inability of IGF-I to stimulate hepatocytes. However, this is not necessarily due to insensitivity of the IGF-IR to IGF-I. For example, serumstarved 3T3 cells do not proliferate in response to IGF-I, yet exhibit IGF-I– dependent IGF-IR phosphorylation.44 Rather, the current data strongly suggest that IGF-IR insensitivity to IGF-I is due to the prevention of IGF-I binding to the IGF-IR by IGFBPs.27 In particular, IGFBP-3 inhibits the proliferation of several cell types.11,28,45 Our current findings of IGF-IR activation by des(1-3)-IGF-I, and surface coating by IGFBP-3, support an inhibitory role for this class of IGFBP in hepatocytes. IGFBP-3 is synthesized by hepatic endothelial cells and Kupffer cells.46,47 Thus, it is likely that IGFBP-3 associates with hepatocytes, as evidenced by the current study. In summary, our findings provide insight into a novel pathway of IGF-IR phosphorylation in hepatocytes. The transactivation of the IGF-IR by EGF and Src provides a mechanism by which the IGF-IR could participate in intracellular signaling leading to mitogenesis. The survival properties of the IGF-IR also may play an important function during liver regeneration. In particular, the regenerating liver is stimulated by several cytokines with pleiotropic activities, notably tumor necrosis factor ␣. Activation of intracellular signaling pathways by the IGF-IR and other survival factors might diminish the apoptotic influences exerted by these cytokines.
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