IGFs stimulate zebrafish cell proliferation by activating MAP kinase ...

Am J Physiol Regulatory Integrative Comp Physiol 280: R1230–R1239, 2001.

IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways KASIANI C. POZIOS,1 JUN DING,1 BRIAN DEGGER,2 ZEE UPTON,3 AND CUNMING DUAN1 Department of Biology, University of Michigan, Ann Arbor, Michigan 48109; 2Cooperative Research Center for Tissue Growth and Repair, Adelaide SA 5000; and 3School of Life Sciences, Queensland University of Technology, Brisbane QLD 4001, Australia

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Received 21 September 2000; accepted in final form 27 November 2000

Pozios, Kasiani C., Jun Ding, Brian Degger, Zee Upton, and Cunming Duan. IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways. Am J Physiol Regulatory Integrative Comp Physiol 280: R1230–R1239, 2001.—Insulin-like growth factor (IGF)-I and -II have been cloned from a number of teleost species, but their cellular actions in fish are poorly defined. In this study, we show that both IGF-I and -II stimulated zebrafish embryonic cell proliferation and DNA synthesis in a concentration-dependent manner, whereas insulin had little mitogenic activity. Affinity cross-linking and immunoblotting studies revealed the presence of IGF receptors with the characteristics of the mammalian type I IGF receptor. Competitive binding assay results indicated that the binding affinities of the zebrafish IGF-I receptors to IGF-I, IGF-II, and insulin are 1.9, 2.6, and ⬎190 nM, indicating that IGF-I and -II bind to the IGF-I receptor(s) with approximately equal high affinity. To further investigate the cellular mechanism of IGF actions, we have studied the effects of IGFs on two major signal transduction pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase). IGFs activated MAPK in zebrafish embryonic cells in a dose-dependent manner. This activation occurred within 5 min of IGF-I stimulation and disappeared after 1 h. IGF-I also caused a concentration-dependent activation of protein kinase B, a downstream target of PI3 kinase, this activation being sustained for several hours. Inhibition of MAPK activation by the MAPK kinase inhibitor PD-98059 inhibited the IGF-I-stimulated DNA synthesis. Similarly, use of the PI3 kinase inhibitor LY-294002 also inhibited IGF-I-stimulated DNA synthesis. When both the MAPK and PI3 kinase pathways were inhibited using a combination of these compounds, the IGF-I-stimulated DNA synthesis was completely negated. These results indicate that both IGF-I and -II are potent mitogens for zebrafish embryonic cells and that activation of both the MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGFs in zebrafish embryonic cells. insulin-like growth factor; insulin-like growth factor I receptor; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; protein kinase B; zebrafish embryos; deoxyribonucleic acid synthesis

THE INSULIN-LIKE GROWTH FACTORS (IGFs), including IGF-I and -II, are a family of single-chain polypeptides struc-

Address for reprint requests and other correspondence: C. Duan, Dept. of Biology, Univ. of Michigan, Natural Science Bldg., Ann Arbor, MI 48109-1048 (E-mail: [email protected]). R1230

turally related to proinsulin. Most of the biological actions of IGFs are exerted through the type I IGF receptor or IGF-I receptor (IGF-IR). As with the insulin receptor, the IGF-IR has a heterotetrameric structure (␣2␤2) with a tyrosine kinase domain in the cytoplasmic portion of the ␤-subunit (8). Despite structural similarities between the receptors and ligands, each receptor binds preferentially to its respective ligand. The IGF-IR binds to IGF-I with a 15- to 20-fold and 100- to 1,000-fold higher affinity than to IGF-II and insulin, respectively (20, 23). Activation of the IGF-IR is linked to two major intracellular signaling pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase) (18), and these have been shown to lead to diverse biological responses ranging from stimulating proliferation, differentiation, migration, and metabolism to inhibition of apoptosis in mammalian systems (31). In mammals, a second transmembrane IGF receptor, the IGF-II/ mannose 6-phosphate receptor, also exists and preferentially binds to IGF-II over IGF-I. Binding of IGF-II to the IGF-II/mannose 6-phosphate receptor has been shown to cause internalization and degradation of IGF-II (20, 28). It has long been established that IGF-I mediates many of the growth-promoting effects of growth hormone (GH) during mammalian postnatal life (20). More recent studies have shown that IGF-I and -II are also essential for fetal growth in mammals, although the actions of IGFs in fetal stages are GH independent (2, 24). Despite these advances, the precise role of various members of the IGF system during vertebrate embryogenesis is not clearly understood. Research in this area has relied heavily on rodent models, but attempts have been hampered by the inaccessibility of the mammalian fetus enclosed in the uterus. During the past decade, there has been a rapid accumulation of knowledge concerning the IGF-signaling system in nonmammalian vertebrates, particularly in bony fish. The sequences of IGF-I cDNAs have been determined in coho salmon, rainbow trout, Atlantic salmon, catfish, carp, seabream, tilapia, the daddy sculpin, and others, The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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IGF SIGNALING AND ZEBRAFISH CELL PROLIFERATION

and their expression has been studied (for review, see Refs. 10, 11, 21, and 32). The IGF-II sequence is now known for salmon, rainbow trout, seabream, barramundi, tilapia, and the daddy sculpin (for reviews, see Refs. 10, 11, 21, and 32). These sequences indicate that the structure of both IGF-I and -II has been highly conserved in bony fish. With the use of 125I-labeled human IGF-I and/or insulin as ligands, the presence of distinct fish IGF-I and insulin receptors has been demonstrated (9, 17, 29, 30), and it has been shown that fish IGF-I and human IGF-I are equally potent in binding to the fish IGF-IR (22). This last finding agrees with functional data showing equal potency of salmon and human IGF-I in stimulation of fish cartilage sulfation and DNA synthesis (3, 27). In addition, partial sequences of IGF-IR genes have been determined in turbot, coho salmon, and rainbow trout (6, 14, 16). The expression of the IGF-IR genes in rainbow trout showed similar developmental and tissue-specific distributions as in mammals and chickens (1, 16, 20) and is consistent with ligand-binding studies in other teleosts (9). Taken together, these findings indicate that the IGF system is highly conserved in teleost fish. We believe that fish models can contribute much to our understanding of the role of the IGF system, particularly in early development. Fish embryos and larva, unlike mammalian embryos that live within the uterus and are dependent on maternal contributions through the placenta, grow freely in water. Hence, the accessibility and rapid development of teleost fish, especially zebrafish, make them well suited for determining the mechanisms by which IGFs act to regulate cell proliferation, differentiation, and apoptosis in early life stages. The transparency of zebrafish embryos provides an additional, immense advantage to their use in developmental studies and makes zebrafish a particularly suitable model system for investigating the mechanisms of IGF actions during early development. As part of our efforts in defining the developmental roles of IGFs, IGF receptors, and IGF-binding proteins, we have studied the mitogenic effects of IGFs in zebrafish embryonic cells (ZF-4) and investigated the underlying intracellular signaling mechanisms that are activated by IGFs. Our results indicate that ZF-4 express the IGF-IRs and their downstream signaling components and that both IGF-I and -II are potent regulators of ZF-4 proliferation. MATERIALS AND METHODS

Materials. All chemicals and reagents were purchased from Sigma (St. Louis, MO), unless noted otherwise. IGF-IR ␤-subunit antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Insulin-receptor substrate (IRS)-1 and 4G10 anti-phosphotyrosine antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Phosphospecific and control antibodies for MAPK, Akt/PKB, and MEK1 inhibitor PD-98059 were purchased from New England Biolabs (Beverly, MA). LY-294002 was purchased from BIOMOL (Plymouth Meeting, PA). Horseradish peroxidaselinked anti-rabbit antibody and rainbow molecular weight markers were from Amersham Life Science (Piscataway, NJ). IGF-I and -II were obtained from GroPep. Fetal bovine serum

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(FBS), antibiotics, Hams’ F12, and DMEM were purchased from Gibco BRL (Gaithersburg, MD). Cell culture. ZF-4 were obtained from American Type Culture Collection (Manassas, VA). The cells were grown in 10-cm dishes (Falcon) in a 1:1 mixture of Hams’ F12 and DMEM with penicillin (100 U/ml), streptomycin (100 ␮g/ml), and 10% FBS at 28.5°C. The medium was changed every third day until the cells became confluent. Before stimulation experiments, medium was changed to serum-free medium (SFM) for 18–24 h. This SFM was then replaced with SFM plus indicated growth factors for various times. Receptor-binding assay. ZF-4 grown to confluence in 12well plates (Falcon) were used for binding assays. The binding assays were performed in HEPES-binding buffer (0.1 M HEPES, 0.12 M NaCl, 5 mM KCl, 1.2 mM MgSO42⫺, 8 mM glucose, and 0.1% BSA, pH 7.8). 125I-labeled IGF-I and unlabeled hormones were used at the indicated concentrations. After incubation at 4°C overnight, the binding buffer and the tracer were removed, and the cells were washed and then solubilized in 0.1% SDS. The radioactivity was counted in a liquid-scintillation counter. Affinity cross-linking study. Confluent ZF-4 in 10-cm plates were washed and then incubated with 125I-labeled IGF-I (1 ⫻ 106 cpm) at 15°C for 3 h in the absence or presence of unlabeled competitor. Affinity cross-linking of monolayer cells with disuccinimidyl suberate was performed according to Parrizas et al. (29, 30). Cell lysates were prepared and analyzed by 10% SDS-PAGE and autoradiography. Western immunoblotting. The cell lysates were separated by SDS-PAGE. After transfer to filters (Immobilon P, 0.45-␮m pore size, Millipore), the membranes were blocked in 3% BSA (Fisher Scientific) in Tris-buffered saline-Tween 20 (TBST). The blots were incubated with a 1:1,000 to 1:5,000 dilution of the indicated antibody in blocking buffer for 1–2 h at room temperature. Blots were then washed with TBST and incubated with a 1:3,000 dilution of horseradish peroxidase-linked anti-rabbit secondary antibody in blocking buffer for 2–3 h, followed by further washing. Enhanced chemiluminescence was performed according to the manufacturer’s instructions (Amersham). Densitometry was performed by scanning the autoradiographs (ScanJet IIcx, Hewlett-Packard) and then analyzing them using Scion Image software. BrdU immunocytochemical staining. To measure the mitogenic activity of IGFs, ZF-4 were plated onto coverslips in 6-well plates and grown to 100% confluency. After growth was arrested by overnight serum starvation, cells were exposed to 20 ␮M 5-bromo-2⬘-deoxyuridine (BrdU) and the desired concentrations of IGF-I and/or inhibitors. After ⬃22 h, cells were washed twice with ice-cold PBS, fixed with 2.0% formaldehyde-0.2% gluteraldehyde in PBS, and then permeablized with acetone-methanol (1:1). The cells were rehydrated in PBS and denatured for 1 h in 2.0 N HCl. Next, the cells were incubated in sodium tetraborate and equilibrated in PBS. The coverslips were then blocked for 2 h in 20% normal goat serum and 0.5% Triton X-100 followed by incubation overnight in a 6 ␮g IgG/ml solution of anti-BrdU. After being washed, the coverslips were exposed to a tetramethylrhodamine isothiocyanate-linked secondary anti-mouse antibody for 2 h (10:1). The coverslips were then washed and exposed for 1 h in darkness to 4⬘,6-diamidino-2-phenylindole at 0.25 mg/ml. The coverslips were washed three times in PBS and mounted onto slides. Cells were counted under a Nikon E600 fluorescence microscope facilitated with the Optronics Camera System. DNA synthesis assay. To determine the rate of DNA synthesis, thymidine-incorporation assays were performed as described previously (13). Briefly, cells were plated onto 96-

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well plates (Falcon) at 50,000 cells/well and incubated for 3–5 days without a medium change. After being rinsed three times with DMEM, the cultures were exposed to 200 ␮l serum-free DMEM/F12 medium containing 1 ␮Ci [3H]thymidine (ICN Biochemicals) and the desired concentrations of IGF-I and/or inhibitor(s). Each treatment was added to triplicate cultures. After ⬃42–44 h, the cells were washed twice with PBS, twice with cold 5% trichloroacetic acid for 10 min at 4°C, and solubilized in 200 ␮l of 0.1 M NaOH-1% SDS at room temperature. The solubilized DNA was harvested for liquid-scintillation counting. The results are expressed as the percent change from the controls. Statistical analysis. One-way ANOVA followed by Fisher’s protected least-significant differences test was used to compare differences between control and test groups using Statview (Abacus concept, Berkeley, CA). Values are means ⫾ SE. RESULTS

IGF-I and -II stimulate ZF-4 proliferation. The mitogenic effects of IGFs were examined by immunocytochemical detection of BrdU incorporation following growth arrest by 24-h serum starvation. As shown in Fig. 1A, BrdU staining was not detected in serumstarved cells, with the exception of rare nuclei. Human IGF-I treatment (100 ng/ml) of growth-arrested cells resulted in a 397 ⫾ 78% increase in the number of BrdU-positive cells over the control. Similar mitogenic activity was observed with human IGF-II (376 ⫾ 65%), suggesting that both IGF-I and -II are potent mitogens for these cells. To further study the mitogenic activity of IGFs, their effects on DNA synthesis were studied by [3H]thymidine-incorporation assays. Exposure of ZF-4 to human IGF-I and -II resulted in a dose-dependent increase in [3H]thymidine incorporation (Fig. 1B), inducing increases of 371 ⫾ 37 and 333 ⫾ 30%, respectively, at the concentration of 500 ng/ml. Human insulin, however, had no effect at concentrations up to 500 ng/ml. To examine the functional conservation of the IGF-I molecule, the effects of human IGF-I, chicken IGF-I, salmon IGF-I, barramundi IGF-I, as well as salmon insulin were compared (Fig. 1C). These results show that human and chicken IGF-I are of approximately equal potency in the zebrafish system compared with that of fish (salmon and barramundi) IGF-I. In

Fig. 1. Insulin-like growth factor (IGF)-I and -II stimulate zebrafish embryonic cells (ZF-4) proliferation. A: effects of IGFs on ZF-4 proliferation. Serum-starved, confluent ZF-4 were exposed to IGF-I or IGF-II (100 ng/ml) in the presence of 5-bromo-2⬘-deoxyuridine (BrdU) (20 ␮M) for 22 h. Cells were fixed and immunostained for BrdU (left: staining proliferating nuclei) and 4⬘,6-diamidino-2-phenylindole (DAPI) (right: staining all nuclei). B: effects of human IGF-I (E), human IGF-II (䊐), and human insulin (‚) on DNA synthesis of cultured ZF-4. Cells grown to confluence were exposed to various concentrations of human IGF-I, human IGF-II, and human insulin in the presence of 1 ␮Ci/well [3H]thymidine. Values are means ⫾ SE of 3 separate experiments; each was performed in triplicate. C: effects of human IGF-I (■), chicken IGF-I (Œ), barramundi IGF-I (), salmon IGF-I (}), and salmon insulin (F) on DNA synthesis of cultured ZF-4. Cells grown to confluence were exposed to various concentrations of IGFs and insulin in the presence of 1 ␮Ci/well [3H]thymidine. Values are means ⫾ SE of 2 separate experiments; each was performed in triplicate.

comparison, human and fish insulin had no significant mitogenic activity in cultured ZF-4. ZF-4 express type I IGF receptors. To determine the cellular mechanisms underlying the IGF mitogenic actions in ZF-4, competitive IGF-binding studies were performed to characterize the IGF receptors. To achieve this, 125I-labeled human IGF-I was incubated with varying concentrations of competing cold human IGF-I, human IGF-II, and human insulin (Fig. 2A). The binding data were analyzed by Scatchard analysis,


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that this receptor(s) binds to IGF-I and -II with an approximately equal high affinity. IGF stimulation activates both the MAPK and PI3 kinase-signaling pathways. To determine the effect of IGF-I stimulation in tyrosine phosphorylation of the IGF-IR and other endogenous proteins, cells were treated with IGF-I (100 ng/ml) for varied exposure times or with different concentrations of IGF-I. As shown in Fig. 3, addition of IGF-I caused tyrosine phosphorylation of several proteins with apparent molecular masses of 42, 96, and 185 kDa, respectively. Immunoblotting analysis using specific antibodies indicated that the 96-kDa protein is the IGF-IR ␤-subunit and that the 42-kDa protein is MAPK. On the basis of the molecular mass and the IGF-I-induced

Fig. 2. Expression of the IGF-I receptors in ZF-4 cells. A: specific IGF-binding site in cultured ZF-4. 125I-labeled human IGF-I was incubated with cultured ZF-4 for 3 h. Unlabeled human IGF-I (■), human IGF-II (Œ), human insulin (F), and salmon insulin (}) in varying concentrations were used to displace the radiolabeled IGF-I. Each point represents the mean of triplicates. B: presence of the IGF-I receptors in cultured ZF-4. 125I-labeled human IGF-I was cross-linked to cultured ZF-4 in the absence (lane 1) or presence of 1 ␮g of human IGF-I (lane 2), human IGF-II (lane 3), human insulin (lane 4), Des(1–3) IGF-I (lane 5), and Leu (1–30) IGF-I (lane 6) as competitor. IGF-IR, IGF-I receptor.

and the results indicated the presence of a single-class, high-affinity binding site. The Kd values for IGF-I, IGF-II, and insulin were 1.9, 2.6, and ⬎190 nM, respectively, showing that IGF-I and -II bind to this receptor with an approximately equal high affinity. Cross-linking of 125I-labeled human IGF-I to cultured ZF-4 resulted in two bands at the size of 125 and 200 kDa, corresponding to the size of the mammalian IGF-IR ␣/␤-complex and ␣-subunit alone, respectively. Consistent with the results obtained in the competitive binding assay, the presence of unlabeled human IGF-I and -II abolished this signal (Fig. 2B, lanes 2 and 3), whereas the addition of human insulin at this concentration had no effect (lane 4). The addition of des(1–3) IGF-I, an IGF analog that binds to the IGF-IR not to IGF-binding proteins (IGFBPs), was able to compete for binding of the radiolabel, whereas addition of Leu27 IGF-I, an IGF analog that binds IGFBPs not the receptor, did not compete (Fig. 2B, lanes 5 and 6). These results indicate that IGF-IR is expressed in ZF-4 and

Fig. 3. IGF-I stimulation leads to tyrosine phosphorylation of several endogenous proteins. A: serum-starved ZF-4 were treated with or without IGF-I at various concentrations for 10 min. The cells were lysed and subjected to SDS-PAGE on a 10% gel. Proteins containing phosphotyrosine were detected with Western immunoblotting with a monoclonal anti-phosphotyrosine antibody. Proteins with increased tyrosine phosphorylation are indicated by the 4. B: serum-starved ZF-4 were treated with or without IGF-I (100 ng/ml) for various time intervals. The cells were lysed and subjected to SDS-PAGE on a 10% gel. Proteins containing phosphotyrosine were detected with Western immunoblotting. Proteins with increased tyrosine phosphorylation are indicated by the 4. IRS-1, insulin-receptor substrate 1; MAPK, mitogen-activated protein kinase.

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Fig. 4. IGF-I stimulation leads to rapid and transient activation of MAPK. A: time-dependent activation of p44/42 MAPK by IGF-I. Serum-starved ZF-4 were treated with or without IGF-I (100 ng/ml) for various periods of time. Lysates were subjected to Western blotting with phospho-specific and control p44/42 MAPK. B: dose-dependent effect of IGF-I in activating p44/42 MAPK. Serum-starved ZF-4 were treated with or without IGF-I at various concentrations for 10 min. Lysates were subjected to Western blotting with the antibodies.

tyrosine phosphorylation property, the 185-kDa protein is likely to be the zebrafish homolog of insulinreceptor substrate 1 (IRS-1). However, this fish protein did not react with an anti-human IRS-1 antibody in Western immunoblotting analysis (data not shown). We next examined the effect of IGF-I in activating the MAPK and PI3 kinase-signaling cascades. The phosphorylation of p44/42 MAPK was examined using phospho-specific and control antibodies. As shown in Fig. 4A, the IGF-I-induced MAPK phosphorylation was observed within 5 min and lasted ⬃1 h. When treated for 10 min, IGF-I induced a dose-dependent phosphorylation of p44/42 MAPK at concentrations ranging from 10 to 250 ng/ml (Fig. 4B). The results of PKB/Akt phosphorylation indicate that the IGF-I-induced PKB/ Akt phosphorylation was observed within 5 min and lasted for at least 6 h (Fig. 5A). This IGF-I-induced increase in the serine phosphorylation of PKB/Akt was concentration dependent at concentrations up to 20

Fig. 5. IGF-I stimulation leads to sustained activation of PKB/Akt. A: time-dependent activation of PKB/Akt by IGF-I. Serumstarved ZF-4 were treated with or without IGF-I (100 ng/ml) for various periods of time. Lysates were subjected to Western blotting with phospho-specific and control Akt/PKB. B: dose-dependent effect of IGF-I in activating PKB/Akt. Serum-starved ZF-4 were treated with or without IGF-I at various concentrations for 10 min. Lysates were subjected to Western blotting with the antibodies.

ng/ml (Fig. 5B). These results suggest that although IGF-I activates both MAPK and PI3 kinase-signaling cascades in ZF-4, the threshold and duration of the IGF-I effects appear to be different. The effect of IGF-I on MAPK activation was transient, and the maximal activation required a relatively high concentration of IGF-I (100 ng/ml). In contrast, IGF-I caused a sustained activation of Akt/PKB, and the maximal activation was seen at 20 ng/ml. The effectiveness of IGFs from various vertebrates in the activation of the MAPK and PI3 kinase pathways in zebrafish cells was also examined. Confluent ZF-4 were exposed to human IGF-I and -II, chicken IGF-I, salmon IGF-I, and barramundi IGF-I at 100 ng/ml for 10 min. The phosphorylation of PKB/Akt and p44/42 MAPK was examined. The results indicate that IGF-I from diverse vertebrate species was able to induce the phosphorylation of these proteins to a similar degree in zebrafish cells (Fig. 6).


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Fig. 6. Effects of various vertebrate IGFs in activating MAPK and PKB/Akt. A: activation of p44/42 MAPK by various vertebrate IGFs. Serum-starved ZF-4 were treated with or without human IGF-I, human IGF-II, chicken IGF-I, salmon IGF-I, and barramundi IGF-I (20 ng/ml) for 10 min. Lysates were subjected to Western blotting with phosphospecific and control p44/42 MAPK. B: activation of PKB/Akt by various vertebrate IGFs across species. Serum-starved ZF-4 were treated with or without human IGF-I, human IGF-II, chicken IGF-I, salmon IGF-I, and barramundi IGF-I (20 ng/ml) for 10 min. Lysates were subjected to Western blotting with phospho-specific and control PKB/Akt.

Activation of both the MAPK and PI3 kinase-signaling pathways is required for IGF-stimulated ZF-4 proliferation. To examine the roles of the MAPK and PI3 kinase pathways in mediating the mitogenic effects of IGFs, the specific inhibitors of MEK and PI3 kinase were used. As shown in Fig. 7A, IGF-I-induced phosphorylation of MAPK was almost completely inhibited by addition of PD-98059 at 40 ␮M. The PI3 kinase inhibitor LY-294002 (20 ␮M) had no effect on MAPK phosphorylation. Likewise, preincubation with LY-294002 at 20 ␮M completely abolished IGF-I-stimulated PKB/AKT phosphorylation, whereas exposure to PD-98059 (40 ␮M) had no such effect (Fig. 7B). These results indicate that the conventional inhibitors of the MAPK and PI3 kinase pathways are effective in zebrafish cells.

The involvement of PI3 kinase and/or MAPK in IGFI-stimulated ZF-4 proliferation was determined by BrdU-staining experiments using growth-arrested cells. As shown in Fig. 8A, treatment with LY-294002 (20 ␮M) or PD-98059 (40 ␮M) partially attenuated IGF-I-stimulated cell proliferation, whereas treatment with both inhibitors at the same time completely abolished IGF-I-induced ZF-4 proliferation. To further investigate the roles of the PI3 kinase and MAPK pathways in mediating the mitogenic effect of IGF-I, thymidine-incorporation assays were also performed. LY-294002 attenuated IGF-I-stimulated thymidine incorporation by 114 ⫾ 8% as well as lowering the basal level to 49 ⫾ 10% of the control group (Fig. 8B). PD98059 also partially inhibited IGF-I-induced thymidine

Fig. 7. Specific inhibition of MAPK and PKB/Akt activation. A: inhibition of IGF-I activation of MAPK by MEK inhibitor PD-98059 (PD). Serum-starved ZF-4 were exposed to media with or without PD (40 ␮M) or LY-294002 (LY; 20 ␮M) for 2 h and then treated with or without IGF-I (100 ng/ml) for 10 min. Lysates were subjected to Western blotting with phospho-specific and control p44/42 MAPK. B: inhibition of IGF-I activation of PKB/Akt by phosphatidylinositol 3-kinase (PI3 kinase) inhibitor LY. Serum-starved ZF-4 were exposed to media with or without PD (40 ␮M) or LY (20 ␮M) for 2 h and then treated with or without IGF-I (100 ng/ml) for 10 min. Lysates were subjected to Western blotting with phospho-specific and control Akt/PKB. SFM, serumfree medium.

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Fig. 8. Inhibition of MAPK and PI3 kinase activation abolishes IGF-I-stimulated ZF-4 proliferation. A: effects of PD and LY. Serum-starved, confluent ZF-4 were exposed to PD (40 ␮M) and LY (20 ␮M) for 2 h and then treated with or without IGF-I (100 ng/ml) in the presence of BrdU (20 ␮M) for 22 h. Cells were fixed and immunostained for BrdU (left: staining proliferating nuclei) and DAPI (right: staining all nuclei). B: effects of PD and LY on IGF-I-stimulated DNA synthesis. Confluent ZF-4 were exposed to media containing isotope and IGF-I (100 ng/ml), IGF-I ⫹ PD (40 ␮M), PD (40 ␮M), IGFI ⫹ LY (20␮M), LY (20 ␮M), and IGFI ⫹ PD (40 ␮M) ⫹ LY (20 ␮M). The values are means ⫾ SE of 3 replicates.

incorporation by 86 ⫾ 4% and lowered basal levels to 25 ⫾ 4% of the SFM control group, whereas addition of both LY-294002 and PD-98059 completely abolished IGF-I-induced thymidine incorporation (Fig. 8B). These results indicate that the activation of both the PI3 kinase and MAPK pathways is required for the mitogenic action of IGF-I in ZF-4. DISCUSSION

In mammals, two types of IGF receptors, type I and type II receptors, have been characterized in addition to the insulin receptor. The biological actions of IGFs are thought to be mediated through interaction with the type I IGF receptor or IGF-IR. The role of the type II or IGF-II/mannose-6-phosphate receptor remains obscure, but comparative studies indicate that chicken and amphibian mannose 6-phosphate receptors do not possess an IGF-binding capacity (4, 7, 36). Therefore, the IGF-II-binding property of this receptor, as well as any of its physiological function with regards to IGF, might have been a later acquisition during evolution. Previous studies in fish have shown the presence of

functional IGF-IR in fish liver and brain, ovaries, skeletal muscle, and in testicular and male germ cells (17, 25, 29, 30). Our study in ZF-4 suggests the presence of one class of IGF receptor(s) with the biochemical characteristics of the mammalian IGF-IR. In competition binding assays, however, the observed binding affinity, IGF-I ⫽ IGF-II 3 insulin, is different from those reported in mammals (5, 15, 19, 37), where IGF-I demonstrated a 15- to 20-fold higher affinity for the receptor than IGF-II. The ligand-binding properties of the IGF-IR in ZF-4 are similar to those of chick IGF-IR (34). The size of the membrane protein affinity labeled with 125I-labeled IGF-I is 125 and 200 kDa, which is comparable to the molecular size of the mammalian IGF-IR ␣/␤-complex and ␣-subunit. Therefore, the IGF receptor expressed in ZF-4 shares many similar characteristics of the mammalian IGF-IR. The signal transduction pathways used by IGF have not been well studied in teleost systems. Studies using mammalian model systems (mostly tumor and transformed cell lines) indicate that one of the earliest steps in signal transduction by the IGF-IR is the phosphor-


ylation of adaptor/docking proteins such as IRS-1 or -2, Shc, Grab2, and Grab10 (23, 35). These molecules then interact with downstream signal transducers and effectors, resulting in activation of the MAPK and PI3 kinase-signaling pathways. Activation of the MAPK pathway is critical for cell proliferation, whereas the PI3 kinase pathway is considered to be important for mediating the metabolic, antiapoptotic, and differentiation actions of IGF-I (23). It should be emphasized that intracellular signaling pathways induced by the IGF-IR are highly cell-type specific. Diploid normal cells like the ZF-4 used in this study, which are untransformed, may respond differently compared with those immortalized cell lines often used for signal transduction studies (12). Indeed, our recent studies using primarily cultured porcine vascular smooth muscle cells suggest that the mitogenic signal of IGF-I is mediated through the PI3 kinase-signaling pathway in these primary cell cultures (12). Therefore, these observations of IGF signal transduction mechanisms made in immortalized mammalian cell lines may not be applicable to ZF-4. In this report, we have studied the effects of IGF-I on MAPK and PI3 kinase activation and investigated the roles of these pathways in mediating IGF-I-stimulated cell growth. Examination of IGF-I-stimulated tyrosine phosphorylation of the IGF-IR and other endogenous proteins revealed the tyrosine phosphorylation of several major proteins including the IGF-IR ␤-subunit, an IRS-1-like protein, and MAPK. Further analyses indicated that both the MAPK and PI3 kinase pathways are activated following IGF-I stimulation in ZF-4. Although IGF-I activates both MAPK and PI3 kinase-signaling cascades in ZF-4, the threshold and duration of the IGF-I effects appear to be different. The effect of IGF-I on MAPK activation was transient (5⬃60 min), and the maximal activation required a relatively high concentration of IGF-I (100 ng/ml). In contrast, IGF-I caused a strong activation of Akt, and the maximal activation was seen at 20 ng/ml. This activation occurred within minutes and was sustained for at least 6 h. We used the selective MEK inhibitor PD-98059 and PI3 kinase inhibitor LY-294002 to examine the role of MAPK and PI3 kinase in mediating the growth signal of IGF-I. Our data suggest that the mitogenic signal of IGF-I is mediated through both the MAPK and PI3 kinase-signaling pathways in cultured ZF-4. Activation of the MAPK-signaling pathway by IGF-I has been shown in a variety of mammalian cell types (20). The importance of MAPK in cell proliferation and gene expression is generally acknowledged. Several targets of this pathway have been defined, including transcription factors such as Elk-1 or AP-1. This provides a common route by which signals from various growth factors and hormones converge at a major regulatory element in the promoters of c-fos and other coregulated genes, the serum response element. In this study, we found that IGF-I caused a transient activation of MAPK in ZF-4, and this activation was nearly completely inhibited by 40 ␮M PD-98059. Specific inhibition of the MAPK activation in these cells significantly

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inhibited, but did not abolish, IGF-I-stimulated DNA synthesis and cell proliferation, suggesting that MAPK activation is critical for IGF-I-induced cell proliferation in these cells. LY-294002 is a compound that specifically inhibits PI3 kinase activity and PKB/Akt activation. Indeed, the use of LY-294002 has facilitated studies establishing roles for PI3 kinase in transducing numerous effects of IGF-I, including regulating metabolism and differentiation and inhibiting apoptosis (35). In ZF-4, LY-294002 at 20 ␮M almost completely inhibited IGF-I-stimulated Akt phosphorylation. At this concentration, LY-294002 significantly inhibited the IGF-I-stimulated DNA synthesis and cell proliferation, indicating that activation of PI3 kinase is required for mediating the growth signal of IGF-I in cultured ZF-4. When both MAPK and PI3 kinase were blocked using the combination of PD-98059 and LY-294002, the IGFI-stimulated DNA synthesis and cell proliferation were completely negated. These results indicate that activation of both MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGF-I in ZF-4. The finding that activation of PI3 kinase, as well as MAPK, is required for the mitogenic action of IGF-I in ZF-4 is yet another example demonstrating that intracellular signaling pathways induced by IGF-IR are likely to be cell-type specific. The PI3 kinase pathway is considered to be important for mediating the metabolic, antiapoptotic, and differentiating actions of IGF-I based on studies done in transformed mammalian cell lines. Clearly, the results of this study indicate that PI3 kinase is also involved in transducing the mitogenic signal of IGF-I in ZF-4. Because these cells are derived from embryos of a teleost species, the different results may be explained by differences between species and/or developmental stages. Several recent studies, however, have implicated PI3 kinase in the induction of mitogenesis by IGF-I in mammalian cells. Milansincic et al. (26) reported that IGF-I strongly activated PI3 kinase in mouse C2C12 myoblasts. In these cells, IGF-I elicited a strong mitogenic response, yet it only had minimal effect on MAPK activity. Likewise, it has been shown that PI3 kinase rather than MAPK activity correlated with IGF-I-induced mitogenesis in early passages of cultured human fibroblasts (33). Moreover, inhibition of PI3 kinase activation by LY-294002 blocked IGF-I-stimulated cell proliferation and DNA synthesis in primary cultures of porcine vascular smooth muscle cells (12). Therefore, it is possible that the PI3 kinase-signaling pathway plays a more important role in transmitting the growth signal of IGF-I than previously recognized. A further aspect of the IGF system that we examined in this study was the functional conservation of IGFs throughout vertebrate evolution. The relative ability of IGFs from varied vertebrate species to activate MAPK and Akt, as well as their mitogenic activities in ZF-4, is highly similar. This finding is consistent with our previous studies showing that fish, chicken, and mammalian IGF-Is are equally potent in stimulating DNA synthesis in cultured human cells (3). Together, these data indicate that the degree of conservation in the

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functionality of the IGF-I molecule in vertebrates is extremely high. In conclusion, the results of this study indicate that IGFs are potent regulators of ZF-4 proliferation. ZF-4 express IGF receptors with characteristics of the mammalian type I IGF receptor, and this receptor has approximately equal binding affinities for IGF-I and -II. The two major signal transduction pathways, MAPK and PI3 kinase, are activated by IGF-I and together induce cell proliferation when stimulated by IGF-I. Further in vivo approaches are now needed to examine how the IGF system, and each of its components, functions to regulate cell growth during early development in zebrafish.

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