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An artificial extracellular matrix created by hepatocyte growth factor fused to IgG-Fc
Biomaterials 31 (2010) 802–809
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An artificial extracellular matrix created by hepatocyte growth factor fused to IgG-Fc Koji Azuma a, Masato Nagaoka b, Chong-Su Cho c, Toshihiro Akaike a, * a
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, USA c Department of Agricultural Biotechnology, Seoul National University, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2009 Accepted 29 September 2009 Available online 20 October 2009
The design of artificial extracellular matrices (ECM) has attracted much attention in tissue engineering and regenerative medicine as well as in molecular biology research. A recombinant hepatocyte growth factor (HGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated as AeHGF-Fc) was constructed and confirmed by Western blot assay. Almost similar amounts of HepG2 cells adhered to AeHGF-Fccoated surface compared to collagen-coated one with large morphological changes. Immobilized AeHGFFc continuously activated Akt in HepG2 cells whereas Akt activation induced by soluble HGF rapidly decreased with time, indicating that immobilized AeHGF-Fc follows different signal transduction pathways compared to soluble HGF. Ó 2009 Published by Elsevier Ltd.
Keywords: Hepatocyte growth factor ECM (extracellular matrix) Cell morphology HepG2 c-Met Akt
1. Introduction The development of an artificial extracellular matrix (ECM) is very important because the ECMs facilitate localization and delivery of cells to specific sites in the body, and they maintain a three-dimensional space for the formation of new tissues with appropriate structure [1]. Also, regulation of cellular behaviors by the ECMs has been a concern in tissue engineering as well as in cell biology research. Recently, progress in recombinant DNA technology has allowed the construction of recombinant protein-based polymers with precisely desined molecular weights, composition, sequences and stereochemistries [2]. Such precise control over molecular structure of the polymer gives similarly fine control over its physicochemical characteristics and biological properties [3]. Also, it enables construction of new tailormade biomaterials with interesting properties important for tissue engineering.
* Corresponding author. Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-57 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: þ81 45 924 5790; fax: þ81 45 924 5815. E-mail address: [email protected] (T. Akaike). 0142-9612/$ – see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2009.09.105
In our previous studies, Nagaoka et al. constructed a fusion protein consisting of E-cadherin extracellular domain and IgG Fc region (E-cad-Fc) to stably adsorb to the cell culture surface. The results indicated that hepatocytes adhered to the E-cad-Fc-coated surface were almost same as collagen-coated one and the adhesion was inhibited by pretreatment of hepatocytes with anti-E-cadherin antibody [4]. They also applied for culture of embryonic stem cells [5]. Interestingly, the adhered stem cells remained separated from each other even in the presence of leukemia inhibitory factor with dendritic morphologies. Furthermore, Ogiwara et al. constructed a fusion protein consisting of epidermal growth factor (EGF) as a cell growth function and IgG Fc region (EGF-Fc) [6,7]. The results showed that 3T3 cells adhered to EGF-Fc-coated surface was similar to collagen-coated one and immobilized EGF-Fc continued to activate MAPK after 4 h without internalization of growth factor. In this study, we aimed to construct hepatocyte growth factor (HGF) fused to IgG-Fc (Activation essential HGF-Fc: AeHGF-Fc) by recombinant genetic technique for application to artificial ECM. The HGF as the pleiotropic growth factor regulates cell survival, morphogenesis, adhesion, migration, breakdown of ECM and angiogenesis by binding with c-Met receptor. Also, HGF and its receptor c-Met play an important role in cancer growth and metastasis [8]. Different intracellular signal transduction will be induced by immobilization of AeHGF-Fc to the polymer surface compared with soluble HGF itself.
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Fig. 1. Cloning and expression of AeHGF-Fc. The cDNA coding for HGF and IgG-Fc region were cloned into the expression vector (A). The fusion protein was detected by Western blotting using an anti-HGF antibody (B) and an anti-mouse IgG antibody (C).
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Fig. 2. Adsorption of AeHGF-Fc on PS dish (A) and adhesion of HepG2 cells on PS dish (B). The amount of adsorbed AeHGF-Fc protein on PS dish was analyzed by ELISA. HepG2 cells adhered to the AeHGF-Fc and collagen-coated dishes after 3 h of incubation. The data indicates means SD of experiments (n ¼ 3).
2. Materials and methods
phospho-Akt antibody (Cell signaling Technology), followed by horseradish peroxidase-conjugated secondary antibodies.
2.1. Construction of AeHGF-Fc
2.4. Cell culture
In this study, the N-terminal hairpin loop, the Kringle 1 and 2 domains of the
a-chain in the HGF (abbreviated as AeHGF domain) were used to construct AeHGFFc because it was not possible to insert whole HGF chains due to big size and AeHGF domain is enough to bind with c-Met [9–11]. To construct AeHGF-Fc, the cDNA that encodes mouse HGF was amplified with KOD plus DNA polymerase (TOYOBO) from the cDNA of mouse fetal liver. Mouse AeHGF domain cDNA was cloned by PCR using 50 -GGA TCC GAA CTG CAA GCA TGA TGT GG-30 and 50 -GCG GCC GCT CTC ATT CAC AGC ACT GTG AGC-30 primers. To generate pRC-AeHGF-Fc vector, the amplified HGF cDNA was introduced into BamH I and Not I recognition sites of the cloning plasmid pGEM-T easy vector (Promega) and the vector containing mouse AeHGF domain cDNA was digested by BamH I and Not I and subcloned into pRC/CMV vector containing mutated mouse Fc fragment cDNA [12]. CHO-K1 cells were transfected with pRC-AeHGF-Fc vector, and highly expressing clone was selected.
2.2. Purification of AeHGF-Fc The obtained fusion protein, AeHGF-Fc, was loaded onto a rProtein A FF column (GE Healthcare). The column was washed with 20 mM phosphate buffer (pH 7.0), and the bound proteins were eluted using 0.1 M sodium citrate (pH 2.7) followed by neutralization with 20% volume of 1.0 M Tris–HCl (pH 9.0). Eluates were dialyzed for 3 days in PBS containing 0.9 mM CaCl2 and 0.9 mM MgCl2.
Human hepatoma cell line HepG2 cells (RIKEN) were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitorogen) containing 10% (v/v) fetal bovine serum (FBS) and antibiotics (penicillin 50 mg/ml and streptomycin 50 mg/ml) under 5% (v/v) CO2 at 37 C. 2.5. Coating of AeHGF-Fc Purified AeHGF-Fc solution was directly added to polystyrene dishes at a final concentration of 10 mg/ml. After incubation at 37 C for 2 h, the dishes were washed with PBS once, plates were incubated for 1 h with 1.0% bovine serum albumin (BSA)/ PBS solution and then cells were seeded. 2.6. ELISA The amount of adsorbed AeHGF-Fc on polystyrene dish was analyzed by ELISA method. Purified AeHGF-Fc solution was directly added to polystyrene dishes at several concentrations. After incubation at 37 C for 2 h, the dishes were washed with PBS once, and plates were incubated for 1 h with 1.0% bovine serum albumin (BSA)/PBS solution to block unspecific interaction. The amount of adsorbed AeHGFFc on polystyrene dish was detected after incubation with peroxidase-conjugated anti-mouse IgG antibody for 1 h. We used o-phenylenediamine (OPD) as a substrate for peroxidase, and we measured the absorbance at 492 nm.
2.3. Detection of AeHGF-Fc by Western blot analysis
2.7. Adhesion assay
The total cellular protein was extracted with lysis buffer (20 mM Tris–HCl (pH. 7.4), 150 mM NaCl, 1% Nonidet P40, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, and proteinase inhibitor). Protein concentration was measured using a DC protein assay kit (Bio-Rad). Equal amount of proteins were separated by SDS-PAGE in reduced conditions. Blots were probed with anti-HGF antibody (R&D Systems), anti-Met (Santa Cruz Biotechnology), anti-phospho-Met antibody (Upstate), anti-p44/42 MAPK antibody (Cell Signaling Technology), anti-phospho-p44/42 MAPK (Cell Signaling Technology), anti-Akt antibody (Cell Signaling Technology) and anti-
HepG2 cells were plated onto AeHGF-Fc- or collagen-coated 96-well plates pretreated with 0.1% (w/v) BSA at 37 C for 2 h. As a negative control, 96-well plates were coated with 10 mg/ml mouse IgG (Jackson Immuno Research Laboratories). After 3 h of incubation in DMEM containing 0.5% (v/v) FBS at 37 C, medium and non-adherent cells were removed, and cells were washed with PBS. Adherent cells were stained with alamar Blue (Biosource International) and absorbance at 570 nm was measured using a microplate reader. The percentage of cell adhesion was normalized against that of cell adhesion onto collagen.
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Fig. 3. Cytoskeleton of adhered HepG2 cells. HepG2 cells were cultured in DMEM containing 0.5% FBS for 3 h in the 24-well plate coated with collagen, coated with collagen in the presence of with HGF, coated with collagen in the presence of with AeHGF-Fc and coated with AeHGF-Fc. a0 , b0 , c0 and d0 are magnified images of the box in a, b, c and d, respectively. HepG2 cells were stained with Alexa Fluor 488 phalloidin to observe for actin. 2.8. Observation of cell cytoskeleton 4
HepG2 cells were seeded at a density of 1.0 10 cells/well into 24-well plates coated with the indicated substrate. After 3, 12, and 24 h of culture, the cell cytoskeleton was observed by the microscopy. For fluorescence staining, cells were fixed with 8% formaldehyde solution (pH 7.0–7.5; Wako Pure Chemical) for 10 min and permeabilized with 0.2% Triton X-100 for 2 min at room temperature. Fixed cells were incubated with Image-iT FX signal enhancer (Invitrogen) for 30 min at room temperature. Actin was visualized using Alexa Fluor 488 phalloidin (Molecular Probes). Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI; Sigma Chemical). Samples were observed by fluorescence microscopy. Each cell contour was traced with a pen tablet (WACOM), and projected area and perimeter of least 20 cells were quantified using Image J. The form factor was calculated as 4P x (area)/(perimeter)2, which is used as an index of the circularity of cell [13]. 2.9. Effect of inhibitors on cell function First, the effect of inhibitors, PD98059 (MEK inhibitor, CALBIOCHEM) or wortmannin (PI3-K inhibitor, Tocris) on MAPKs and PI3-K/Akt pathways was investigated
by Western blotting analysis. Then, HepG2 cells were pretreated with 50 mM PD98059 and/or 1 mM wortmannin for 2 h. After recovering the HepG2 cells, these cells were cultured on immobilized AeHGF-Fc for 2 h. The status of active form of ERK and Akt was determined by Western blot analysis.
3. Results 3.1. Construction of AeHGF-Fc The expression vector for the fusion protein AeHGF-Fc was prepared by fusing the IgG-Fc region to the C-terminal of AeHGF after transfection in CHO-K1 cells as shown in Fig. 1A. The AeHGF-Fc was purified by the conditioned media of the selected transfected CHO-K1 cell clone. The purified fusion protein was detected by Western blotting using anti-HGF antibody and anti-mouse IgG
K. Azuma et al. / Biomaterials 31 (2010) 802–809
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Fig. 4. Quntification of cell morphological changes. HepG2 cells were cultured in DMEM containing 0.5% FBS for 3, 12 and 24 h in the 24-well plate coated with collagen, coated with collagen in the presence of HGF, coated with collagen in the presence of AeHGF-Fc and coated with immobilized AeHGF-Fc. Cell area (A), perimeter (B), and form factor (C) were plotted against culture time. At least 20 cells on each substrate were analyzed. The data indicates means SD (n ¼ 3), *: p < 0.001 versus cells cultured on collagen for 3h, #: p < 0.001 vs. cells cultured on collagen with HGF for 3h, x: p < 0.001 vs. cells cultured on collagen with AeHGF-Fc for 3h.
antibody as shown in Fig. 1B and C. Also, the fusion protein existed as a homodimer via the hinge region of Fc fragment (data not shown). 3.2. Adsorption of AeHGF-Fc on polystyrene (PS) dish First, the amount of AeHGF-Fc adsorption onto a polystyrene dish was estimated by ELISA. Adsorption of AeHGF-Fc to the
polystyrene dish increased with a dose-dependent manner through Langmuir’s adsorption isotherm mechanism (Fig. 2A). When AeHGF-Fc was added at a concentration of approximately 10 mg/ml, adsorbed AeHGF-Fc on polystyrene dish was saturated. To prepare the immobilized AeHGF-Fc-coated dishes, AeHGF-Fc solution was directly added to polystyrene dishes at a concentration of 10 mg/ml.
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Fig. 5. Effect of inhibitors on morphological changes of the cells by immobilized AeHGF-Fc. The serum-starved HepG2 cells were pretreated with the specific inhibitor PD98059 (50 mM) and/or PI3-K inhibitor wortmannin (1 mM) for 2 h. After recovering the HepG2 cells, these cells were cultured on immobilized AeHGF-Fc for 2 h. a0 , b0 , c0 and d0 are magnifications of the box in a, b, c and d, respectively. HepG2 cells were stained with Alexa Fluor 488 phalloidin to observe actin fiber (A). Cell area (B), perimeter (C), and form factor (D) were plotted. At least 20 cells on each substrate were analyzed. The data indicates means SD (n ¼ 3), *: p < 0.001 versus cells without inhibitors.
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Fig. 6. Time course of c-Met, ERK and Akt activation HepG2 cells were seeded onto AeHGF-Fc- or collagen-coated dishes and cultured in DMEM containing 0.5% FBS for various times. HepG2 cells cultured in collagen-coated dishes were stimulated with or without 100 nM of HGF or AeHGF-Fc. Lysates were subjected to Western blotting.
3.3. Adhesion of HepG2 cells onto AeHGF-Fc-coated PS dish Adhesion of HepG2 cells was performed to check whether the cells could adhere to the AeHGF-Fc-coated PS dish. As shown in Fig. 2B, almost 90% of cells adhered to AeHGF-Fc-coated surface whereas only 10% of cells adhered to IgG-coated one, suggesting that the cell adhesion is mediated by AeHGF region not by the Fc region. It is suggested that obtained AeHGF-Fc has the ability of binding with c-Met because the AeHGF-Fc contains the N-terminal hairpin loop, the Kringle 1 and 2 domains, which are essential domains for binding with c-Met [9–11]. 3.4. Effect of immobilized AeHGF-Fc on cell cytoskeleton It is already reported that cell growth and migration are closely related to reorganization of the cell cytoskeleton. Fig. 3 shows the cytoskeleton of HepG2 cells adhered to collagen-coated, collagencoated in the presence of HGF, collagen-coated in the presence of AeHGF-Fc and AeHGF-Fc-coated dishes. The results indicated that more morphological changes of HepG2 cells adhered to the AeHGFFc-coated dish with more actin fibers were observed than another ones, suggesting that immobilized AeHGF-Fc induce different signal transduction pathways than the soluble HGF as already
previously reported [4–7,14]. To quantify morphological changes of adhered HepG2 cells against time, area, perimeter and form factor of the adhered cells were calculated in the Fig. 4. The results indicated that the form factor as the index of circularity of cells [13] adhered to the AeHGF-Fc surface was smaller than another ones and was decreased with an increase of time. To determine whether the morphological changes of HepG2 cells adhered to the immobilized AeHGF-Fc surface is involved in the intracellular signal transduction, we examined the effect of these inhibitors on cell cytoskeleton. Fig. 5A shows the cytoskeleton of HepG2 cells adhered to the AeHGF-Fc-coated, the AeHGF-Fc-coated in the presence of PD98059, the AeHGF-Fc-coated in the presence of wortmannin and the AeHGF-Fc coated dishes in the presence of PD98059 and wortmannin. The results showed that treatment with PD98059 and/or wortmannin decreases morphological changes of HepG2 cells adhered to the AeHGF-Fc-coated dish. To quantify morphological changes of HepG2 cells, area, perimeter and form factor of the adhered cells were estimated in the Figs. 5B–D. The results indicated that the form factor of cells adhered to the AeHGFFc surface with PD98059 or wortmannin was bigger than without PD98059 and wortmannin. In addition, pretreatment with PD98059 and wortmannin decreased morphological changes more than another ones, suggesting that both phosphorylation of ERK1/2
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and Akt induced by the immobilized AeHGF-Fc were involved in morphological changes of HepG2 cells. 3.5. Activation of c-Met, ERK and Akt by immobilized AeHGF-Fc HGF induces cell proliferation, morphogenesis, motogenesis and anti-apoptosis by binding with c-Met receptor [15]. It has been reported that activated ERK by HGF is involved in proliferation in HepG2 cells [16–18]. Also, protein kinase B (PKB)/Akt as a prosurvival intracellular signaling molecule is known to be activated in the beta cell by HGF [19]. Fig. 6 shows the time course of c-Met (A), ERK (B) and Akt (C) activation in HepG2 cells induced by collagencoated, collagen-coated in the presence of HGF, collagen-coated in the presence of AeHGF-Fc and AeHGF-Fc-coated surfaces. The results indicated that the immobilized AeHGF-Fc transduced a signal to the cells through the activated c-Met. In addition, immobilized AeHGF-Fc still activated Akt after 24 h, whereas the activation of Akt in the cells adhered to collagen-coated surfaces in the presence of HGF or AeHGF-Fc rapidly decreased with time, suggesting that Akt activation induced by the immobilized AeHGFFc in the cells was continuous without diffusion and internalization. However, activation of c-Met and ERK in the cells adhered to the immobilized AeHGF-Fc was not much different from another ones. The actin reorganization is initiated by intercellular signaling mechanisms that includes activation of the ERK and PI3-K [20]. To test whether activated ERK and Akt in HepG2 cells cultured on AeHGF-Fc-coated dish is involved in cell shape changes, we check effect of inhibitors such as PD98059 as MEK inhibitor and wortmannin as PI3-K inhibitor. First, we evaluated effect of the inhibitors on the phosphorylation of ERK1/2 and Akt induced by the immobilized AeHGF-Fc. The results revealed that wortmannin and/or PD98059 suppressed the immobilized AeHGF-Fc-induced ERK1/2 and/or Akt activation (Fig. 7). 4. Discussion In this study, we constructed genetically engineered-recombinant HGF (AeHGF-Fc) because the HGF regulates cell survival, morphogenesis, adhesion, migration, breakdown of ECM and angiogenesis by binding with c-Met receptor. The construction of the AeHGF-Fc was confirmed by Western blotting assay although the fusion protein existed as a homodimer via the hinge region of Fc fragment. The AeHGF-Fc was adsorbed to the PS dish with a dose-dependent manner through Langmuir’s adsorption isotherm mechanism. Also, it is suggested that the AeHGF-Fc stably adsorb to the PS dish with the exposure of HGF domain in the outer with the spatial orientation due to the hydrophobic property of the IgG domain [6,7]. Almost 90% of HepG2 cells adhered to the AeHGF-Fc-coated PS surface whereas only 10% of the cells adhered to the IgG-coated one, indication of specific interaction between HGF and c-Met receptor in the cells although the mechanism of the specific interaction should be cleared in more detail. Also, more morphological changes of HepG2 cells adhered to the AeHGF-Fc-coated PS dish occurred with more actin fibers compared with another ones, suggestion of different signal transduction pathways between immobilized growth factor and soluble one [4–7,14], and some information about mechanism on invasion of tumor cells because invasion cells is closely related to reorganization of the cell cytoskeleton and activated ERK and Akt pathway through phosphorylation of c-Met is also associated with morphological changes of the cells [21–25]. As expected, the immobilized AeHGF-Fc induced more longterm activation of Akt than soluble one because cell growth was enhanced by immobilized growth factors due to the inhibition of down-regulation of the receptors without internalization of growth
Fig. 7. Effect of inhibitors on activation of ERK and Akt by immobilized AeHGF-Fc. The serum-starved HepG2 cells were pretreated with the specific inhibitor PD98059 (50 mM) and/or PI3-K inhibitor wortmannin (1 mM) for 2 h. After recovering the HepG2 cells, these cells were cultured on immobilized AeHGF-Fc for 2 h. The status of active form of ERK(A) and Akt(B) was determined by Western blot analysis.
factors and prevention of lateral diffusion of the activated receptors in the plane of the cell membrane [26]. However, activation of c-Met and ERK in the cells adhered to the immobilized AeHGF-Fc was not much different from soluble one although the exact mechanism is not clear in this moment. 5. Conclusion The fusion protein AeHGF-Fc successfully constructed by the recombinant genetic technique had the ability of binding with c-Met and retained activity of HGF. Immobilized AeHGF-Fc had higher cell adhesion with large morphological change and induced longer Akt activation with different signal transduction pathway compared to soluble HGF. The AeHGF-Fc will be expected to use as a new artificial ECM. Acknowledgements This research is supported by grants from the Ministry of Education, Sports, Science and Technology of Japan. This work is
K. Azuma et al. / Biomaterials 31 (2010) 802–809
financially supported by the Grant-in-Aid for 21st Century COE program. Appendix Figure with essential color and discrimination. Figs. 3 and 5 of this article may be difficult to interpret in black and white. The full color images can be found in the online version, at doi:10.1016/j. biomaterials.2009.09.105. References [1] Bouhadir KH, Mooney DJ. In vitro and in vivo models for the reconstruction of intracellular signaling. Ann N Y Acad Sci 1998;842:188–94. [2] Meyer DE, Chilkoti A. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules 2002;3:357–67. [3] Ghandehari H, Cappello J. Genetic engineering of protein-based polymers: potential in controlled drug delivery. Pharm Res 1998;15:813–5. [4] Nagaoka M, Ise H, Akaike T. Immobilized E-cadherin model can enhance cell attachment and differentiation of primary hepatocytes but not proliferation. Biotechnol Lett 2002;24:1857–62. [5] Nagaoka M, Koshimizu U, Yuasa S, Hattori F, Chen H, Tanaka T, et al. E-cadherin coated plates maintain pluripotent ES cells without colony formation. PLoS One 2006;1:e15. [6] Ogiwara K, Nagaoka M, Cho CS, Akaike T. Construction of a novel extracellular matrix using a new genetically engineered epidermal growth factor fused to IgG-Fc. Biotechnol Lett 2005;27:1633–7. [7] Ogiwara K, Nagaoka M, Cho CS, Akaike T. Effect of photo-immobilization of epidermal growth factor on the cellular behaviors. Biochem Biophys Res Commun 2006;345:255–9. [8] Lesko E, Majka M. The biological role of HGF-MET axis in tumor growth and development of metastasis. Front Biosci 2008;13:1271–80. [9] Okigaki M, Komada M, Uehara Y, Miyazawa K, Kitamura N. Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains. Biochemistry 1992;31:9555–61. [10] Matsumoto K, Takehara T, Inoue H, Hagiya M, Shimizu S, Nakamura T. Deletion of kringle domains or the N-terminal hairpin structure in hepatocyte growth factor results in marked decreases in related biological activities. Biochem Biophys Res Commun 1991;181:691–9. [11] Lokker NA, Mark MR, Luis EA, Bennett GL, Robbins KA, Baker JB, et al. Structure-function analysis of hepatocyte growth factor: identification of variants
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24]
[25]
[26]
809
that lack mitogenic activity yet retain high affinity receptor binding. EMBO J 1992;11:2503–10. Nagaoka M, Akaike T. Single amino acid substitution in the mouse IgG1 Fc region induces drastic enhancement of the affinity to protein A. Protein Eng 2003;16:243–5. Sirois E, Charara J, Ruel J, Dussault JC, Gagnon P, Doillon CJ. Endothelial cells exposed to erythrocytes under shear stress: an in vitro study. Biomaterials 1998;19:1925–34. Ito Y, Zheng J, Imanishi Y, Yonezawa K, Kasuga M. Protein-free cell culture on an artificial substrate with covalently immobilized insulin. Proc Natl Acad Sci U S A 1996;93:3598–601. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor receptor as the c-met protooncogene product. Science 1991;251:802–4. Kondo A, Hirayama N, Sugito Y, Shono M, Tanaka T, Kitamura N. Coupling of Grb2 to Gab1 mediates hepatocyte growth factor-induced high intensity ERK signal required for inhibition of HepG2 hepatoma cell proliferation. J Biol Chem 2008;283:1428–36. Tsukada Y, Miyazawa K, Kitamura N. High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2. J Biol Chem 2001;276:40968–76. Yang H, Magilnick N, Noureddin M, Mato JM, Lu SC. Effect of hepatocyte growth factor on methionine adenosyltransferase genes and growth is cell density-dependent in HepG2 cells. J Cell Physiol 2007;210:766–73. ˜ a A. Improving islet transplantation Fiaschi-Taesch N, Stewart AF, Garcia-Ocan by gene delivery of hepatocyte growth factor (HGF) and its downstream target, protein kinase B (PKB)/Akt. Cell Biochem Biophys 2007;48:191–9. Svoboda KK, Orlow DL, Chu CL, Reenstra WR. ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent. Anat Rec 1999;254:348–59. Sipeki S, Bander E, Buday L, Farkas G, Ba´csy E, Ways DK, et al. Phospatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinae activation associated with hepatocyte growth factor-induced cell scattering. Cell Signal 1999;11:885–90. Birchmeier C, Gherardi E. Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase. Trends Cell Biol. 1998;8:404–10. Royal I, Fournier TM, Park M. Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis. J Cell Physiol 1997;173: 196–201. Royal I, Park M. Hepatocyte growth factor-induced scatter of madin-darby canine kidney cells requires phosphatidylinositol 3-kinase. J Biol Chem 1995;270:27780–7. Khwaja A, Lehmann K, Marte BM, Downward J. Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway. J Biol Chem 1998;273:18793–801. Ito Y, Chen G, Imanishi Y, Morooka T, Nishida E, Okabayashi Y, et al. Differential control of cellular gene expression by diffusible and non-diffusible EGF. J Biochem 2001;129:733–7.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
An artificial extracellular matrix created by hepatocyte growth factor fused to IgG-Fc Koji Azuma a, Masato Nagaoka b, Chong-Su Cho c, Toshihiro Akaike a, * a
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, USA c Department of Agricultural Biotechnology, Seoul National University, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 September 2009 Accepted 29 September 2009 Available online 20 October 2009
The design of artificial extracellular matrices (ECM) has attracted much attention in tissue engineering and regenerative medicine as well as in molecular biology research. A recombinant hepatocyte growth factor (HGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated as AeHGF-Fc) was constructed and confirmed by Western blot assay. Almost similar amounts of HepG2 cells adhered to AeHGF-Fccoated surface compared to collagen-coated one with large morphological changes. Immobilized AeHGFFc continuously activated Akt in HepG2 cells whereas Akt activation induced by soluble HGF rapidly decreased with time, indicating that immobilized AeHGF-Fc follows different signal transduction pathways compared to soluble HGF. Ó 2009 Published by Elsevier Ltd.
Keywords: Hepatocyte growth factor ECM (extracellular matrix) Cell morphology HepG2 c-Met Akt
1. Introduction The development of an artificial extracellular matrix (ECM) is very important because the ECMs facilitate localization and delivery of cells to specific sites in the body, and they maintain a three-dimensional space for the formation of new tissues with appropriate structure [1]. Also, regulation of cellular behaviors by the ECMs has been a concern in tissue engineering as well as in cell biology research. Recently, progress in recombinant DNA technology has allowed the construction of recombinant protein-based polymers with precisely desined molecular weights, composition, sequences and stereochemistries [2]. Such precise control over molecular structure of the polymer gives similarly fine control over its physicochemical characteristics and biological properties [3]. Also, it enables construction of new tailormade biomaterials with interesting properties important for tissue engineering.
* Corresponding author. Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-57 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: þ81 45 924 5790; fax: þ81 45 924 5815. E-mail address: [email protected] (T. Akaike). 0142-9612/$ – see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2009.09.105
In our previous studies, Nagaoka et al. constructed a fusion protein consisting of E-cadherin extracellular domain and IgG Fc region (E-cad-Fc) to stably adsorb to the cell culture surface. The results indicated that hepatocytes adhered to the E-cad-Fc-coated surface were almost same as collagen-coated one and the adhesion was inhibited by pretreatment of hepatocytes with anti-E-cadherin antibody [4]. They also applied for culture of embryonic stem cells [5]. Interestingly, the adhered stem cells remained separated from each other even in the presence of leukemia inhibitory factor with dendritic morphologies. Furthermore, Ogiwara et al. constructed a fusion protein consisting of epidermal growth factor (EGF) as a cell growth function and IgG Fc region (EGF-Fc) [6,7]. The results showed that 3T3 cells adhered to EGF-Fc-coated surface was similar to collagen-coated one and immobilized EGF-Fc continued to activate MAPK after 4 h without internalization of growth factor. In this study, we aimed to construct hepatocyte growth factor (HGF) fused to IgG-Fc (Activation essential HGF-Fc: AeHGF-Fc) by recombinant genetic technique for application to artificial ECM. The HGF as the pleiotropic growth factor regulates cell survival, morphogenesis, adhesion, migration, breakdown of ECM and angiogenesis by binding with c-Met receptor. Also, HGF and its receptor c-Met play an important role in cancer growth and metastasis [8]. Different intracellular signal transduction will be induced by immobilization of AeHGF-Fc to the polymer surface compared with soluble HGF itself.
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Fig. 2. Adsorption of AeHGF-Fc on PS dish (A) and adhesion of HepG2 cells on PS dish (B). The amount of adsorbed AeHGF-Fc protein on PS dish was analyzed by ELISA. HepG2 cells adhered to the AeHGF-Fc and collagen-coated dishes after 3 h of incubation. The data indicates means SD of experiments (n ¼ 3).
2. Materials and methods
phospho-Akt antibody (Cell signaling Technology), followed by horseradish peroxidase-conjugated secondary antibodies.
2.1. Construction of AeHGF-Fc
2.4. Cell culture
In this study, the N-terminal hairpin loop, the Kringle 1 and 2 domains of the
a-chain in the HGF (abbreviated as AeHGF domain) were used to construct AeHGFFc because it was not possible to insert whole HGF chains due to big size and AeHGF domain is enough to bind with c-Met [9–11]. To construct AeHGF-Fc, the cDNA that encodes mouse HGF was amplified with KOD plus DNA polymerase (TOYOBO) from the cDNA of mouse fetal liver. Mouse AeHGF domain cDNA was cloned by PCR using 50 -GGA TCC GAA CTG CAA GCA TGA TGT GG-30 and 50 -GCG GCC GCT CTC ATT CAC AGC ACT GTG AGC-30 primers. To generate pRC-AeHGF-Fc vector, the amplified HGF cDNA was introduced into BamH I and Not I recognition sites of the cloning plasmid pGEM-T easy vector (Promega) and the vector containing mouse AeHGF domain cDNA was digested by BamH I and Not I and subcloned into pRC/CMV vector containing mutated mouse Fc fragment cDNA [12]. CHO-K1 cells were transfected with pRC-AeHGF-Fc vector, and highly expressing clone was selected.
2.2. Purification of AeHGF-Fc The obtained fusion protein, AeHGF-Fc, was loaded onto a rProtein A FF column (GE Healthcare). The column was washed with 20 mM phosphate buffer (pH 7.0), and the bound proteins were eluted using 0.1 M sodium citrate (pH 2.7) followed by neutralization with 20% volume of 1.0 M Tris–HCl (pH 9.0). Eluates were dialyzed for 3 days in PBS containing 0.9 mM CaCl2 and 0.9 mM MgCl2.
Human hepatoma cell line HepG2 cells (RIKEN) were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitorogen) containing 10% (v/v) fetal bovine serum (FBS) and antibiotics (penicillin 50 mg/ml and streptomycin 50 mg/ml) under 5% (v/v) CO2 at 37 C. 2.5. Coating of AeHGF-Fc Purified AeHGF-Fc solution was directly added to polystyrene dishes at a final concentration of 10 mg/ml. After incubation at 37 C for 2 h, the dishes were washed with PBS once, plates were incubated for 1 h with 1.0% bovine serum albumin (BSA)/ PBS solution and then cells were seeded. 2.6. ELISA The amount of adsorbed AeHGF-Fc on polystyrene dish was analyzed by ELISA method. Purified AeHGF-Fc solution was directly added to polystyrene dishes at several concentrations. After incubation at 37 C for 2 h, the dishes were washed with PBS once, and plates were incubated for 1 h with 1.0% bovine serum albumin (BSA)/PBS solution to block unspecific interaction. The amount of adsorbed AeHGFFc on polystyrene dish was detected after incubation with peroxidase-conjugated anti-mouse IgG antibody for 1 h. We used o-phenylenediamine (OPD) as a substrate for peroxidase, and we measured the absorbance at 492 nm.
2.3. Detection of AeHGF-Fc by Western blot analysis
2.7. Adhesion assay
The total cellular protein was extracted with lysis buffer (20 mM Tris–HCl (pH. 7.4), 150 mM NaCl, 1% Nonidet P40, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, and proteinase inhibitor). Protein concentration was measured using a DC protein assay kit (Bio-Rad). Equal amount of proteins were separated by SDS-PAGE in reduced conditions. Blots were probed with anti-HGF antibody (R&D Systems), anti-Met (Santa Cruz Biotechnology), anti-phospho-Met antibody (Upstate), anti-p44/42 MAPK antibody (Cell Signaling Technology), anti-phospho-p44/42 MAPK (Cell Signaling Technology), anti-Akt antibody (Cell Signaling Technology) and anti-
HepG2 cells were plated onto AeHGF-Fc- or collagen-coated 96-well plates pretreated with 0.1% (w/v) BSA at 37 C for 2 h. As a negative control, 96-well plates were coated with 10 mg/ml mouse IgG (Jackson Immuno Research Laboratories). After 3 h of incubation in DMEM containing 0.5% (v/v) FBS at 37 C, medium and non-adherent cells were removed, and cells were washed with PBS. Adherent cells were stained with alamar Blue (Biosource International) and absorbance at 570 nm was measured using a microplate reader. The percentage of cell adhesion was normalized against that of cell adhesion onto collagen.
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Fig. 3. Cytoskeleton of adhered HepG2 cells. HepG2 cells were cultured in DMEM containing 0.5% FBS for 3 h in the 24-well plate coated with collagen, coated with collagen in the presence of with HGF, coated with collagen in the presence of with AeHGF-Fc and coated with AeHGF-Fc. a0 , b0 , c0 and d0 are magnified images of the box in a, b, c and d, respectively. HepG2 cells were stained with Alexa Fluor 488 phalloidin to observe for actin. 2.8. Observation of cell cytoskeleton 4
HepG2 cells were seeded at a density of 1.0 10 cells/well into 24-well plates coated with the indicated substrate. After 3, 12, and 24 h of culture, the cell cytoskeleton was observed by the microscopy. For fluorescence staining, cells were fixed with 8% formaldehyde solution (pH 7.0–7.5; Wako Pure Chemical) for 10 min and permeabilized with 0.2% Triton X-100 for 2 min at room temperature. Fixed cells were incubated with Image-iT FX signal enhancer (Invitrogen) for 30 min at room temperature. Actin was visualized using Alexa Fluor 488 phalloidin (Molecular Probes). Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI; Sigma Chemical). Samples were observed by fluorescence microscopy. Each cell contour was traced with a pen tablet (WACOM), and projected area and perimeter of least 20 cells were quantified using Image J. The form factor was calculated as 4P x (area)/(perimeter)2, which is used as an index of the circularity of cell [13]. 2.9. Effect of inhibitors on cell function First, the effect of inhibitors, PD98059 (MEK inhibitor, CALBIOCHEM) or wortmannin (PI3-K inhibitor, Tocris) on MAPKs and PI3-K/Akt pathways was investigated
by Western blotting analysis. Then, HepG2 cells were pretreated with 50 mM PD98059 and/or 1 mM wortmannin for 2 h. After recovering the HepG2 cells, these cells were cultured on immobilized AeHGF-Fc for 2 h. The status of active form of ERK and Akt was determined by Western blot analysis.
3. Results 3.1. Construction of AeHGF-Fc The expression vector for the fusion protein AeHGF-Fc was prepared by fusing the IgG-Fc region to the C-terminal of AeHGF after transfection in CHO-K1 cells as shown in Fig. 1A. The AeHGF-Fc was purified by the conditioned media of the selected transfected CHO-K1 cell clone. The purified fusion protein was detected by Western blotting using anti-HGF antibody and anti-mouse IgG
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Fig. 4. Quntification of cell morphological changes. HepG2 cells were cultured in DMEM containing 0.5% FBS for 3, 12 and 24 h in the 24-well plate coated with collagen, coated with collagen in the presence of HGF, coated with collagen in the presence of AeHGF-Fc and coated with immobilized AeHGF-Fc. Cell area (A), perimeter (B), and form factor (C) were plotted against culture time. At least 20 cells on each substrate were analyzed. The data indicates means SD (n ¼ 3), *: p < 0.001 versus cells cultured on collagen for 3h, #: p < 0.001 vs. cells cultured on collagen with HGF for 3h, x: p < 0.001 vs. cells cultured on collagen with AeHGF-Fc for 3h.
antibody as shown in Fig. 1B and C. Also, the fusion protein existed as a homodimer via the hinge region of Fc fragment (data not shown). 3.2. Adsorption of AeHGF-Fc on polystyrene (PS) dish First, the amount of AeHGF-Fc adsorption onto a polystyrene dish was estimated by ELISA. Adsorption of AeHGF-Fc to the
polystyrene dish increased with a dose-dependent manner through Langmuir’s adsorption isotherm mechanism (Fig. 2A). When AeHGF-Fc was added at a concentration of approximately 10 mg/ml, adsorbed AeHGF-Fc on polystyrene dish was saturated. To prepare the immobilized AeHGF-Fc-coated dishes, AeHGF-Fc solution was directly added to polystyrene dishes at a concentration of 10 mg/ml.
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Fig. 5. Effect of inhibitors on morphological changes of the cells by immobilized AeHGF-Fc. The serum-starved HepG2 cells were pretreated with the specific inhibitor PD98059 (50 mM) and/or PI3-K inhibitor wortmannin (1 mM) for 2 h. After recovering the HepG2 cells, these cells were cultured on immobilized AeHGF-Fc for 2 h. a0 , b0 , c0 and d0 are magnifications of the box in a, b, c and d, respectively. HepG2 cells were stained with Alexa Fluor 488 phalloidin to observe actin fiber (A). Cell area (B), perimeter (C), and form factor (D) were plotted. At least 20 cells on each substrate were analyzed. The data indicates means SD (n ¼ 3), *: p < 0.001 versus cells without inhibitors.
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Fig. 6. Time course of c-Met, ERK and Akt activation HepG2 cells were seeded onto AeHGF-Fc- or collagen-coated dishes and cultured in DMEM containing 0.5% FBS for various times. HepG2 cells cultured in collagen-coated dishes were stimulated with or without 100 nM of HGF or AeHGF-Fc. Lysates were subjected to Western blotting.
3.3. Adhesion of HepG2 cells onto AeHGF-Fc-coated PS dish Adhesion of HepG2 cells was performed to check whether the cells could adhere to the AeHGF-Fc-coated PS dish. As shown in Fig. 2B, almost 90% of cells adhered to AeHGF-Fc-coated surface whereas only 10% of cells adhered to IgG-coated one, suggesting that the cell adhesion is mediated by AeHGF region not by the Fc region. It is suggested that obtained AeHGF-Fc has the ability of binding with c-Met because the AeHGF-Fc contains the N-terminal hairpin loop, the Kringle 1 and 2 domains, which are essential domains for binding with c-Met [9–11]. 3.4. Effect of immobilized AeHGF-Fc on cell cytoskeleton It is already reported that cell growth and migration are closely related to reorganization of the cell cytoskeleton. Fig. 3 shows the cytoskeleton of HepG2 cells adhered to collagen-coated, collagencoated in the presence of HGF, collagen-coated in the presence of AeHGF-Fc and AeHGF-Fc-coated dishes. The results indicated that more morphological changes of HepG2 cells adhered to the AeHGFFc-coated dish with more actin fibers were observed than another ones, suggesting that immobilized AeHGF-Fc induce different signal transduction pathways than the soluble HGF as already
previously reported [4–7,14]. To quantify morphological changes of adhered HepG2 cells against time, area, perimeter and form factor of the adhered cells were calculated in the Fig. 4. The results indicated that the form factor as the index of circularity of cells [13] adhered to the AeHGF-Fc surface was smaller than another ones and was decreased with an increase of time. To determine whether the morphological changes of HepG2 cells adhered to the immobilized AeHGF-Fc surface is involved in the intracellular signal transduction, we examined the effect of these inhibitors on cell cytoskeleton. Fig. 5A shows the cytoskeleton of HepG2 cells adhered to the AeHGF-Fc-coated, the AeHGF-Fc-coated in the presence of PD98059, the AeHGF-Fc-coated in the presence of wortmannin and the AeHGF-Fc coated dishes in the presence of PD98059 and wortmannin. The results showed that treatment with PD98059 and/or wortmannin decreases morphological changes of HepG2 cells adhered to the AeHGF-Fc-coated dish. To quantify morphological changes of HepG2 cells, area, perimeter and form factor of the adhered cells were estimated in the Figs. 5B–D. The results indicated that the form factor of cells adhered to the AeHGFFc surface with PD98059 or wortmannin was bigger than without PD98059 and wortmannin. In addition, pretreatment with PD98059 and wortmannin decreased morphological changes more than another ones, suggesting that both phosphorylation of ERK1/2
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and Akt induced by the immobilized AeHGF-Fc were involved in morphological changes of HepG2 cells. 3.5. Activation of c-Met, ERK and Akt by immobilized AeHGF-Fc HGF induces cell proliferation, morphogenesis, motogenesis and anti-apoptosis by binding with c-Met receptor [15]. It has been reported that activated ERK by HGF is involved in proliferation in HepG2 cells [16–18]. Also, protein kinase B (PKB)/Akt as a prosurvival intracellular signaling molecule is known to be activated in the beta cell by HGF [19]. Fig. 6 shows the time course of c-Met (A), ERK (B) and Akt (C) activation in HepG2 cells induced by collagencoated, collagen-coated in the presence of HGF, collagen-coated in the presence of AeHGF-Fc and AeHGF-Fc-coated surfaces. The results indicated that the immobilized AeHGF-Fc transduced a signal to the cells through the activated c-Met. In addition, immobilized AeHGF-Fc still activated Akt after 24 h, whereas the activation of Akt in the cells adhered to collagen-coated surfaces in the presence of HGF or AeHGF-Fc rapidly decreased with time, suggesting that Akt activation induced by the immobilized AeHGFFc in the cells was continuous without diffusion and internalization. However, activation of c-Met and ERK in the cells adhered to the immobilized AeHGF-Fc was not much different from another ones. The actin reorganization is initiated by intercellular signaling mechanisms that includes activation of the ERK and PI3-K [20]. To test whether activated ERK and Akt in HepG2 cells cultured on AeHGF-Fc-coated dish is involved in cell shape changes, we check effect of inhibitors such as PD98059 as MEK inhibitor and wortmannin as PI3-K inhibitor. First, we evaluated effect of the inhibitors on the phosphorylation of ERK1/2 and Akt induced by the immobilized AeHGF-Fc. The results revealed that wortmannin and/or PD98059 suppressed the immobilized AeHGF-Fc-induced ERK1/2 and/or Akt activation (Fig. 7). 4. Discussion In this study, we constructed genetically engineered-recombinant HGF (AeHGF-Fc) because the HGF regulates cell survival, morphogenesis, adhesion, migration, breakdown of ECM and angiogenesis by binding with c-Met receptor. The construction of the AeHGF-Fc was confirmed by Western blotting assay although the fusion protein existed as a homodimer via the hinge region of Fc fragment. The AeHGF-Fc was adsorbed to the PS dish with a dose-dependent manner through Langmuir’s adsorption isotherm mechanism. Also, it is suggested that the AeHGF-Fc stably adsorb to the PS dish with the exposure of HGF domain in the outer with the spatial orientation due to the hydrophobic property of the IgG domain [6,7]. Almost 90% of HepG2 cells adhered to the AeHGF-Fc-coated PS surface whereas only 10% of the cells adhered to the IgG-coated one, indication of specific interaction between HGF and c-Met receptor in the cells although the mechanism of the specific interaction should be cleared in more detail. Also, more morphological changes of HepG2 cells adhered to the AeHGF-Fc-coated PS dish occurred with more actin fibers compared with another ones, suggestion of different signal transduction pathways between immobilized growth factor and soluble one [4–7,14], and some information about mechanism on invasion of tumor cells because invasion cells is closely related to reorganization of the cell cytoskeleton and activated ERK and Akt pathway through phosphorylation of c-Met is also associated with morphological changes of the cells [21–25]. As expected, the immobilized AeHGF-Fc induced more longterm activation of Akt than soluble one because cell growth was enhanced by immobilized growth factors due to the inhibition of down-regulation of the receptors without internalization of growth
Fig. 7. Effect of inhibitors on activation of ERK and Akt by immobilized AeHGF-Fc. The serum-starved HepG2 cells were pretreated with the specific inhibitor PD98059 (50 mM) and/or PI3-K inhibitor wortmannin (1 mM) for 2 h. After recovering the HepG2 cells, these cells were cultured on immobilized AeHGF-Fc for 2 h. The status of active form of ERK(A) and Akt(B) was determined by Western blot analysis.
factors and prevention of lateral diffusion of the activated receptors in the plane of the cell membrane [26]. However, activation of c-Met and ERK in the cells adhered to the immobilized AeHGF-Fc was not much different from soluble one although the exact mechanism is not clear in this moment. 5. Conclusion The fusion protein AeHGF-Fc successfully constructed by the recombinant genetic technique had the ability of binding with c-Met and retained activity of HGF. Immobilized AeHGF-Fc had higher cell adhesion with large morphological change and induced longer Akt activation with different signal transduction pathway compared to soluble HGF. The AeHGF-Fc will be expected to use as a new artificial ECM. Acknowledgements This research is supported by grants from the Ministry of Education, Sports, Science and Technology of Japan. This work is
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financially supported by the Grant-in-Aid for 21st Century COE program. Appendix Figure with essential color and discrimination. Figs. 3 and 5 of this article may be difficult to interpret in black and white. The full color images can be found in the online version, at doi:10.1016/j. biomaterials.2009.09.105. References [1] Bouhadir KH, Mooney DJ. In vitro and in vivo models for the reconstruction of intracellular signaling. Ann N Y Acad Sci 1998;842:188–94. [2] Meyer DE, Chilkoti A. Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system. Biomacromolecules 2002;3:357–67. [3] Ghandehari H, Cappello J. Genetic engineering of protein-based polymers: potential in controlled drug delivery. Pharm Res 1998;15:813–5. [4] Nagaoka M, Ise H, Akaike T. Immobilized E-cadherin model can enhance cell attachment and differentiation of primary hepatocytes but not proliferation. Biotechnol Lett 2002;24:1857–62. [5] Nagaoka M, Koshimizu U, Yuasa S, Hattori F, Chen H, Tanaka T, et al. E-cadherin coated plates maintain pluripotent ES cells without colony formation. PLoS One 2006;1:e15. [6] Ogiwara K, Nagaoka M, Cho CS, Akaike T. Construction of a novel extracellular matrix using a new genetically engineered epidermal growth factor fused to IgG-Fc. Biotechnol Lett 2005;27:1633–7. [7] Ogiwara K, Nagaoka M, Cho CS, Akaike T. Effect of photo-immobilization of epidermal growth factor on the cellular behaviors. Biochem Biophys Res Commun 2006;345:255–9. [8] Lesko E, Majka M. The biological role of HGF-MET axis in tumor growth and development of metastasis. Front Biosci 2008;13:1271–80. [9] Okigaki M, Komada M, Uehara Y, Miyazawa K, Kitamura N. Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains. Biochemistry 1992;31:9555–61. [10] Matsumoto K, Takehara T, Inoue H, Hagiya M, Shimizu S, Nakamura T. Deletion of kringle domains or the N-terminal hairpin structure in hepatocyte growth factor results in marked decreases in related biological activities. Biochem Biophys Res Commun 1991;181:691–9. [11] Lokker NA, Mark MR, Luis EA, Bennett GL, Robbins KA, Baker JB, et al. Structure-function analysis of hepatocyte growth factor: identification of variants
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24]
[25]
[26]
809
that lack mitogenic activity yet retain high affinity receptor binding. EMBO J 1992;11:2503–10. Nagaoka M, Akaike T. Single amino acid substitution in the mouse IgG1 Fc region induces drastic enhancement of the affinity to protein A. Protein Eng 2003;16:243–5. Sirois E, Charara J, Ruel J, Dussault JC, Gagnon P, Doillon CJ. Endothelial cells exposed to erythrocytes under shear stress: an in vitro study. Biomaterials 1998;19:1925–34. Ito Y, Zheng J, Imanishi Y, Yonezawa K, Kasuga M. Protein-free cell culture on an artificial substrate with covalently immobilized insulin. Proc Natl Acad Sci U S A 1996;93:3598–601. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor receptor as the c-met protooncogene product. Science 1991;251:802–4. Kondo A, Hirayama N, Sugito Y, Shono M, Tanaka T, Kitamura N. Coupling of Grb2 to Gab1 mediates hepatocyte growth factor-induced high intensity ERK signal required for inhibition of HepG2 hepatoma cell proliferation. J Biol Chem 2008;283:1428–36. Tsukada Y, Miyazawa K, Kitamura N. High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2. J Biol Chem 2001;276:40968–76. Yang H, Magilnick N, Noureddin M, Mato JM, Lu SC. Effect of hepatocyte growth factor on methionine adenosyltransferase genes and growth is cell density-dependent in HepG2 cells. J Cell Physiol 2007;210:766–73. ˜ a A. Improving islet transplantation Fiaschi-Taesch N, Stewart AF, Garcia-Ocan by gene delivery of hepatocyte growth factor (HGF) and its downstream target, protein kinase B (PKB)/Akt. Cell Biochem Biophys 2007;48:191–9. Svoboda KK, Orlow DL, Chu CL, Reenstra WR. ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent. Anat Rec 1999;254:348–59. Sipeki S, Bander E, Buday L, Farkas G, Ba´csy E, Ways DK, et al. Phospatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinae activation associated with hepatocyte growth factor-induced cell scattering. Cell Signal 1999;11:885–90. Birchmeier C, Gherardi E. Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase. Trends Cell Biol. 1998;8:404–10. Royal I, Fournier TM, Park M. Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis. J Cell Physiol 1997;173: 196–201. Royal I, Park M. Hepatocyte growth factor-induced scatter of madin-darby canine kidney cells requires phosphatidylinositol 3-kinase. J Biol Chem 1995;270:27780–7. Khwaja A, Lehmann K, Marte BM, Downward J. Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway. J Biol Chem 1998;273:18793–801. Ito Y, Chen G, Imanishi Y, Morooka T, Nishida E, Okabayashi Y, et al. Differential control of cellular gene expression by diffusible and non-diffusible EGF. J Biochem 2001;129:733–7.