Stimulation of the Mitogen-activated Protein Kinase Cascade and Tyrosine Phosphorylation of the Epidermal Growth Factor Receptor by Hepatopoietin

Abstract

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor, which specifically stimulates proliferation of cultured primary hepatocytes in vitro and liver regeneration after liver partial hepatectomy in vivo. Recently, the identification of the mitogenic effect of HPO on hepatoma cell lines and the existence of HPO-specific receptors indicate that HPO acts via its specific cell surface receptor. However, the molecular mechanism of HPO action is not fully elucidated. In this report, we examined the signal transduction events induced by HPO in hepatoma cell line (HepG2). Our results demonstrated that HPO induces phosphorylation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase (MAPK) in a rapid and transient manner. HPO stimulates tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we observed that both MAPK activation and the mitogenic effect of HPO on HepG2 cells were completely blocked by AG1478, a specific inhibitor of EGFR tyrosine kinase activity. However, the effects of HPO were not antagonized by an EGFR-blocking antibody, mAb528, which blocks the interaction between epidermal growth factor and EGFR, indicating that stimulation of tyrosine phosphorylation of EGFR by HPO was not mediated by epidermal growth factor. In contrast, genistein, a general tyrosine kinase inhibitor, significantly attenuated the tyrosine phosphorylation of EGFR in response to HPO. In conclusion, our results suggest that tyrosine phosphorylation of EGFR may play a critical role in MAPK activation and mitogenic stimulation by HPO. Hepatopoietin (HPO) is a novel human hepatotrophic growth factor, which specifically stimulates proliferation of cultured primary hepatocytes in vitro and liver regeneration after liver partial hepatectomy in vivo. Recently, the identification of the mitogenic effect of HPO on hepatoma cell lines and the existence of HPO-specific receptors indicate that HPO acts via its specific cell surface receptor. However, the molecular mechanism of HPO action is not fully elucidated. In this report, we examined the signal transduction events induced by HPO in hepatoma cell line (HepG2). Our results demonstrated that HPO induces phosphorylation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase (MAPK) in a rapid and transient manner. HPO stimulates tyrosine phosphorylation of epidermal growth factor receptor (EGFR). Furthermore, we observed that both MAPK activation and the mitogenic effect of HPO on HepG2 cells were completely blocked by AG1478, a specific inhibitor of EGFR tyrosine kinase activity. However, the effects of HPO were not antagonized by an EGFR-blocking antibody, mAb528, which blocks the interaction between epidermal growth factor and EGFR, indicating that stimulation of tyrosine phosphorylation of EGFR by HPO was not mediated by epidermal growth factor. In contrast, genistein, a general tyrosine kinase inhibitor, significantly attenuated the tyrosine phosphorylation of EGFR in response to HPO. In conclusion, our results suggest that tyrosine phosphorylation of EGFR may play a critical role in MAPK activation and mitogenic stimulation by HPO. hepatopoietin epidermal growth factor hepatocyte growth factor insulin-like growth factor-1 transformation growth factor mitogen-activated protein kinase epidermal growth factor receptor mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Hepatopoietin (HPO)1 is a novel human hepatotrophic growth factor, an orthologue of rat augmenter of liver regeneration or hepatic stimulator substance (1He F.C. Wu C. Chin. Sci. Bull. 1997; 42: 1505-1511Crossref Google Scholar). In 1975, LaBrecque and Pesh (2LaBrecque D.R. Pesh L.A. J. Physiol. 1975; 248: 273-278Crossref PubMed Scopus (184) Google Scholar) first reported that in the livers of weanling rats or partially hepatectomized rats, there existed a polypeptide, named hepatic stimulator substance, that could specifically stimulate DNA synthesis of hepatic cells. The existence of hepatic stimulator substance-related activities has been reported in other species including mice, cows, dogs, pigs, and humans (3LaBrecque D.R. Am. J. Gastroenterol. 1994; 89 (suppl.): 90-96Google Scholar). Hagiya et al. (4Hagiya M. Francavilla A. Polimeno L. Ihara I. Sakai H. Seki T. Shimonishi M. Porter K.A. Starzl T.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8142-8146Crossref PubMed Scopus (179) Google Scholar) cloned the cDNA of rat augmenter of liver regeneration, which is the same as rat hepatic stimulator substance. Subsequently, Giorda et al. (5Giorda R. Hagiya M. Seki T. Mol. Med. 1996; 2: 97-108Crossref PubMed Google Scholar) and Yang et al. (6Yang X.M. Xie L. Qiu Z.H. Wu C. He F.C. Sci. China Ser. C Life Sci. 1997; 6: 642-647Crossref Scopus (22) Google Scholar) cloned the cDNA of human augmenter of liver regeneration or HPO by screening the cDNA library of human fetal liver. HPO encodes a novel protein with no sequence similarity to any known growth regulator. Interestingly, HPO is highly related to the yeast ERV (essential for respiration and viability) gene products. However, the functional relevance of HPO and ERV is currently unclear (7Lisowsky T. Weinstat-Saslow D.L. Barton N. Reeders S.T. Schneider M.C. Genomics. 1995; 29: 690-697Crossref PubMed Scopus (55) Google Scholar). Yang et al. (8,9) demonstrated that the recombinant human HPO stimulated proliferation of hepatocytes as well as hepatoma cells in vitro. HPO also promotes regeneration and recovery of damaged hepatocytes and rescues acute hepatic failurein vivo (8Yang X.M. Hu Z.Y. Xie L. Wu C. He F.C. Acta Physiol. Sin. 1997; 49: 557-561PubMed Google Scholar, 9Yang X.M Xie L. He H. Wei H.D. He F.C. Acta Biochim. Biophys. Sin. 1997; 29: 72-77Google Scholar). Thus, these observations support the contention that HPO is a hepatotrophic growth factor. Unlike other typical growth factors, HPO was discovered in the cytosol of liver parenchymal cells and was produced in an autocrined way (rather than in a paracrined way from mesenchymal cells or in an endocrined way from other glands) during liver regeneration or organogenesis (10LaBrecque D.R. Dig. Dis. Sci. 1991; 36: 669-673Crossref PubMed Scopus (42) Google Scholar). In addition, the effects of HPO are known to be liver-specific. HPO specifically stimulates proliferation of cultured primary hepatocytes in vitro and enhances liver regeneration after liver partial hepatectomy in vivo. HPO displays no significant effect on the proliferation of non-hepatocytes or tumor cell lines derived from tissues other than liver (10LaBrecque D.R. Dig. Dis. Sci. 1991; 36: 669-673Crossref PubMed Scopus (42) Google Scholar). These unique characteristics distinguish HPO from various well known hepatic stimulators, such as insulin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and transformation growth factor (TGF-α), which can stimulate proliferation of a wide variety of cell types (11Fausto N. Lairo A.D. Webber E.M. FASEB J. 1995; 9: 1527-1536Crossref PubMed Scopus (569) Google Scholar). Because of the very specific effect of HPO, the mechanism of HPO action might be intriguing and different from other known growth factors. Recently, we characterized the mitogenic effect of HPO on hepatoma cell lines and demonstrated the existence of HPO receptors on the membranes of these cells (12Wang G. Yang X.M. Zhang Y. Wang Q.M. Chen H.P. Wei H.D. Xing G.C. Xie L. Hu Z.Y. Zhang C.G. Fang D.C. Wu C. He F.C. J. Biol. Chem. 1999; 274: 11469-11472Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Furthermore, we showed that HPO acts as an autocrine growth factor to maintain the autonomous growth of hepatoma cell lines in vitro. These findings suggest that HPO binds a specific receptor on the cell membrane and triggers the signal transduction pathway leading to cell proliferation. Considering the importance of the mitogen-activated protein kinase (MAPK) pathway in cell growth, we investigated activation of the MAPK pathway by HPO. EGF is also an important growth factor in liver regeneration (13Michalopoulos G.K. Defrances M. Science. 1997; 276: 60-66Crossref PubMed Scopus (2874) Google Scholar) and hepatoma progression in vivo (14Yamaguchi K. Carr B.I. Nalesnik M.A. J. Surg. Oncol. 1995; 58: 240-245Crossref PubMed Scopus (49) Google Scholar). The autocrine loop of TGF-α/EGFR plays an important role in supporting autonomous growth of hepatoma cells in vitro (15Yang E.B. Wang D.F. Mack P. Cheng L.Y. Biochem. Mol. Biol. Int. 1996; 38: 813-820PubMed Google Scholar). In addition to activation by its ligands, such as EGF and TGF-α, EGFR could act as a downstream mediator in various signaling pathways via a novel ligand-independent pathway. In some tumor cell lines, EGFR is essential for a wide variety of mitogens to stimulate the MAPK pathway and cell proliferation (16Hackel P.O. Zwick E. Prenzel N. Ullrich A. Curr. Opin. Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (545) Google Scholar). In this study, we demonstrated that EGFR might play an important role for the mitogenic signaling triggered by HPO in hepatoma cell lines. HepG2 (a hepatocellular carcinoma cell line) and GLC-82 (a lung cancer cell line) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% bovine calf serum (Hyclone). HPO open reading frame (375 base pairs) (6Yang X.M. Xie L. Qiu Z.H. Wu C. He F.C. Sci. China Ser. C Life Sci. 1997; 6: 642-647Crossref Scopus (22) Google Scholar) was subcloned into expression vector pBV220 to create vector pBV220-HPO. The vector containing HPO cDNA was transformed into Escherichia coli strain JM109. A single colony of JM109 containing pBV220-HPO to LB (5 ml) was seeded and cultured overnight at 37 °C with vigorous shaking. The culture was diluted 2:100 into fresh LB and incubated for 3 h at 30 °C; then expression of HPO protein was induced by heating (42 °C, 5 h). The bacteria cells were collected by centrifugation (4000 rpm, 10 min) and lysated with a sonicator. The sonicated cells were spun at 10,000 × g for 5 min; then the supernatant was discarded, and the inclusion body pellet was washed with 20 mm Tris-HCl (pH 8.0), 0.5% Triton X-100, 4m urea. The washed inclusion bodies were dissolved in solubilization buffer (8 m urea, 1 mm EDTA in 20 mm Tris-HCl, pH 8.0) and then centrifuged at 10,000 × g for 20 min. The supernatant was diluted to the protein concentration of 10 mg/ml. 5 ml of supernatant of the sample was loaded on the Superdex 75 prep grade 26/60 column (Amersham Pharmacia Biotech) equilibrated with the solubilization buffer. The column was eluted at 2 ml/min. The purity of the proteins of peak II was analyzed by silver-stained SDS-polyacrylamide gel electrophoresis. The purified protein was refolded by dilution in refolding buffer (1 mm reduced glutathione, 0.5 mm oxidized glutathione in 20 mm Tris-HCl, pH 8.0). The refolded protein was dialyzed against 20 mm Tris-HCl (pH 8.0) completely; then the solution was spun at 10,000 × gfor 20 min, and the supernatant was collected. EGF, IGF-1, tyrphostin AG1478, and genistein were from Sigma. Antibodies against active MAP kinase and pan-extracellular signal-regulated kinase were from Promega Corporation. Peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Jackson ImmunoResearch Laboratories, Inc. EGFR, IGF-1 receptor, and HGF receptor antibodies and Protein A/G-agarose were from Santa Cruz Biotechnology. Antibody mAb528 was from NeoMarkers. Phospho-tyrosine monoclonal antibody P-Tyr-100 was from BioLabs. Enhanced Chemiluminescense (ECL) kit and Hybond-P membrane were from Amersham Pharmacia Biotech. Cells were rinsed three times with ice-cold phosphate-buffered saline and lysed in 50 mm HEPES pH 7.4, 1% Nonidet P-40, 100 mm NaCl, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 5 mm sodiumo-vanadate, 20 mm NaF, 10 mm sodium pyrophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin for 20 min at 4 °C. The lysate was centrifuged at 12,000 rpm for 30 min. For detecting HGF receptor, IGF-1 receptor, and EGFR, the supernatant was collected and used in Western blot directly. For detecting the phospho-EGFR, the supernatant was incubated with anti-EGFR antibody for 2 h at 4 °C, followed by incubation with Protein A-Sepharose overnight at 4 °C. The immunoprecipitates were collected by centrifugation and washed by radioimmune precipitation buffer three times. The lysate or immunoprecipitate was fractionated via 10% SDS-polyacrylamide gel electrophoresis. The proteins were blotted onto Hybond-P membranes. The membranes were first blocked by incubation in TTBS containing 3% BSA overnight at 4 °C, then incubated sequentially with primary antibody and peroxidase-conjugated secondary antibody, and detected using an ECL kit. Finally, the various Western blotting bands were scanned by GS-710 calibrated imaging densitometer (Bio-Rad) and analyzed by Quantity OneTMsoftware (Bio-Rad, PhotoShop 4.0). Activation of the MAPK pathway is a key event in cell growth regulation. Both MEK and MAPK are activated by phosphorylation of conserved residues in the activation loop of the kinase domains. Activities of endogenous MEK and MAPK can be indirectly determined by Western blot with phosphospecific antibodies. We tested whether MAPK is stimulated in HepG2 cells by HPO. Fig. 1 indicates that HPO induces phosphorylation of MEK and MAPK in serum-starved HepG2 cells. The increase of phosphorylation appeared in a rapid and transient manner. MEK phosphorylation was at a maximum 5 min after HPO treatment and declined to the basal level at 30 min. Similarly, phosphorylation of MAPK reached the peak at 10 min and dropped to the basal level at 30 min after HPO stimulation. The kinetics of MEK and MAPK induced by HPO is very similar to those activated by other growth factors (17Chaudhary L.R. Avioli L.V. Biochem. Biophys. Res. Commun. 1997; 238: 134-139Crossref PubMed Scopus (70) Google Scholar, 18Dabrowski A. Groblewski G.E. Scher C. Guan K.L. Williams D. Am. J. Physiol. 1997; 273: G1472-G1479Crossref PubMed Google Scholar). The above data demonstrate that HPO activates the MAPK signaling pathway in HepG2 cells. EGFR is activated not only by EGF but also by a wide variety of mitogens. We examined whether HPO stimulates tyrosine phosphorylation of EGFR in HepG2 cells. As shown in Fig. 2, HPO induces tyrosine phosphorylation of EGFR but has no effect on the tyrosine phosphorylation of HGF receptor (c-MET) nor on IGF-1 receptor. The kinetics of HPO action on the tyrosine phosphorylation of EGFR parallels that of the phosphorylation of MEK and MAPK, suggesting that phosphorylation of EGFR may play a role in the activation of MEK and MAPK in response to HPO. The level of phosphorylated EGFR induced by HPO is approximately one-third of that induced by EGF (Fig.2 B). The specificity of phosphorylation of EGFR triggered by HPO is demonstrated by the observation that only HPO and EGF, but neither HGF nor IGF-1, stimulate tyrosine phosphorylation of EGFR (Fig.2 A). To determine the significance of EGFR in HPO signaling, the effect of tyrphostin AG1478 was tested on MAPK activation by HPO. Tyrphostin AG1478 is a specific inhibitor of EGFR tyrosine kinase activity (19Levitki A. Gazit A. Science. 1995; 267: 1782-1787Crossref PubMed Scopus (1616) Google Scholar). Fig. 3indicates that the MAPK activation triggered by both HPO and EGF were completely blocked by AG1478. In contrast, IGF-1-induced MAPK activation was not unaffected. Furthermore, the effect of HPO is more sensitive to the AG1478 than is the effect of EGF (data not shown). This could be due to the fact that EGF induced a much stronger activation of EGFR than did HPO (Fig. 2 A). Our data show that EGFR is a prerequisite of the HPO signaling cascade leading to MAPK activation and that the role of EGFR in HPO signaling depends on its intrinsic AG1478-sensitive tyrosine kinase activity.Figure 1Activation of MEK and MAPK by HPO. HepG2 cells were cultured without serum for 24 h prior to stimulation with 50 ng/ml recombinant human HPO for the indicated times. Cell lysates (50 μg) were fractionated by 10% SDS-polyacrylamide gel electrophoresis, transfected to polyvinylidene difluoride membranes, and probed with antibodies against phospho-MEK (A) or phospho-MAPK (B). The quantification data were obtained by scanning Western blot bands. Bars, standard deviation of triplicated samples (C).View Large Image Figure ViewerDownload (PPT)Figure 3EGFR inactivation inhibited MAPK activation mediated by HPO. cells were without AG1478 (A) or with AG1478 for 10 min stimulation with the indicated for 5 min. The cell lysates were analyzed by antibodies against phospho-MAPK or Large Image Figure ViewerDownload HPO was reported to stimulate DNA synthesis of HepG2 cells. To the role of EGFR in HPO signaling, we the mitogenic of HPO in the or of AG1478. DNA synthesis was determined by The results in Fig. 4 show that AG1478 completely blocked the of DNA synthesis triggered by EGF and HPO, indicating that EGFR is essential for the mitogenic effect of HPO. these results we that the intrinsic tyrosine kinase activity of AG1478-sensitive kinase is a prerequisite of the HPO signaling leading to both MAPK activation and mitogenic was demonstrated that the GLC-82 cell line lung cancer not the specific receptor of HPO (12Wang G. Yang X.M. Zhang Y. Wang Q.M. Chen H.P. Wei H.D. Xing G.C. Xie L. Hu Z.Y. Zhang C.G. Fang D.C. Wu C. He F.C. J. Biol. Chem. 1999; 274: 11469-11472Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To the role of the specific receptor of HPO in HPO signaling, GLC-82 cells were to whether the specific HPO receptor is for the activation of EGFR and MAPK triggered by HPO. The results in Fig. 5 indicate that HPO no effect on the phosphorylation of EGFR nor MAPK in GLC-82 EGF stimulated phosphorylation of both EGFR and These data suggest that the specific receptor of HPO might be for the activation of EGFR and MAPK during HPO has been demonstrated that HPO has its specific receptor that is different from the receptors of EGF, TGF-α, and on the HepG2 EGF, TGF-α, and no effect to the cell surface (12Wang G. Yang X.M. Zhang Y. Wang Q.M. Chen H.P. Wei H.D. Xing G.C. Xie L. Hu Z.Y. Zhang C.G. Fang D.C. Wu C. He F.C. J. Biol. Chem. 1999; 274: 11469-11472Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In vitro indicate no interaction of HPO with the receptors of EGF, TGF-α, and the of EGFR tyrosine phosphorylation by HPO may be a ligand-independent the of tyrosine phosphorylation of EGFR by HPO might be different from a To this the specific antibody mAb528 against EGFR, which has been to the Cell Res. PubMed Scopus (55) Google was Our results show that mAb528 no effect on the tyrosine phosphorylation of EGFR triggered by HPO, completely blocked the tyrosine phosphorylation triggered by EGF The above results suggest that the tyrosine phosphorylation of EGFR triggered by HPO is of The effect of HPO on EGFR is not due to a of HPO to The results in 5 and indicate that HPO binds its receptor and indirectly induces activation of a tyrosine and EGFR tyrosine is a inhibitor of tyrosine (19Levitki A. Gazit A. Science. 1995; 267: 1782-1787Crossref PubMed Scopus (1616) Google Scholar). To we the effect of genistein on EGFR phosphorylation triggered by HPO and The results show that in the of genistein, tyrosine phosphorylation of EGFR was induced and by EGF and HPO, In genistein significantly attenuated the effect of HPO on EGFR phosphorylation approximately at a (5 and completely blocked the effect of HPO at 10 However, genistein no significant effect on EGFR phosphorylation induced by EGFR at the same (5 and 10 and a effect at These results indicate that HPO and EGF different to stimulate the tyrosine phosphorylation of We that HPO may a tyrosine which is different from EGFR and more sensitive to genistein Activation of this tyrosine kinase is to play an important role in HPO signaling genistein completely blocked the HPO effect on the tyrosine phosphorylation of the role of HPO as a stimulator of hepatocyte proliferation in liver regeneration has been investigated the the molecular of HPO action are The MAPK pathway is well demonstrated to be an important pathway in liver regeneration, which was activated by some hepatic growth factors, including the three important EGF, TGF-α, and HGF (11Fausto N. Lairo A.D. Webber E.M. FASEB J. 1995; 9: 1527-1536Crossref PubMed Scopus (569) Google Scholar). mitogens effect in the of the cell including an in which cells to proliferation and a in which hepatocytes and proliferation FASEB J. 1996; PubMed Scopus Google Scholar). However, HPO to act at the and stimulates DNA synthesis of those hepatocytes that the (3LaBrecque D.R. Am. J. Gastroenterol. 1994; 89 (suppl.): 90-96Google Scholar). our results show that MAPK is also activated by HPO. However, in the of characteristics of HPO with those is to that the HPO signaling leading to MAPK activation might be by a mechanism different from other hepatocyte mitogens. and more that EGFR acts not only as a receptor for but also as a mediator of signaling T. C. G. H. Res. 1999; Google Scholar, T. J. Wu J. J. Biol. Chem. 273: Full Text Full Text PDF PubMed Scopus Google Scholar). A wide variety of including some of receptors such as H. C. Ullrich A. 1996; PubMed Scopus Google and with receptors J. Res. 1996; Google stimulate cell growth via active EGFR in a ligand-independent pathway. In this study, we demonstrate that HPO triggers MAPK activation and proliferation in HepG2 cells of tyrosine phosphorylation of Furthermore, our data suggest that activation of EGFR an HPO-specific receptor but not the of HPO or EGF to The characteristics of EGFR activation by HPO are similar to those in an Our findings suggest that HPO binds its receptor and then triggers a tyrosine to EGFR or that HPO the phosphorylation of The of to genistein of EGFR activation induced by HPO and EGF this HPO is of phosphorylation of EGFR, HPO the of EGF For EGF, HPO the phosphorylation of and of in HepG2 cells (data not and stimulate growth of various tissues and cells other than liver and This indicates the of signaling pathways between HPO and This the first data the signal transduction pathway of HPO. We demonstrated that HPO stimulates the activation of EGFR and Furthermore, our results show that tyrosine phosphorylation of EGFR is important for the effect of HPO on activation of MAPK and stimulation of cell proliferation. These data important for the of HPO action and the between HPO and other hepatocyte of the molecular of HPO action be for our of liver regeneration, and Considering that the HPO receptor has not been cloned the significance of EGFR in HPO signaling some on the way to the HPO receptor.