[CANCER RESEARCH 60, 2263–2272, April 15, 2000] Insulin-like Growth Factor I Receptor Signaling in Differentiation of Neuronal H19-7 Cells 1 Andrea Morrione, Gaetano Romano, Magali Navarro, Krzysztof Reiss, Barbara Valentinis, Michael Dews, Eva Eves, Marsha Rich Rosner, and Renato Baserga 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [A. M., G. R., M. N., K. R., B. V., M. D., R. B.], and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 [E. E., M. R. R.] ABSTRACT The type I insulin-like growth factor receptor (IGF-IR) is known to send two seemingly contradictory signals inducing either cell proliferation or cell differentiation, depending on cell type and/or conditions. H19-7 cells are rat hippocampal neuronal cells immortalized by a temperature- sensitive SV40 large T antigen that grow at 34°C in epidermal growth factor or serum but differentiate at 39°C when induced by basic fibroblast growth factor. At 39°C, expression of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I-dependent differentiation. We have investigated the domains of the IGF-IR required for differenti- ation of H19-7 cells. The tyrosine 950 residue and serines 1280 –1283 in the COOH terminus of the receptor are required for IGF-I-induced differen- tiation at 39°C, although they are dispensable for IGF-I-mediated growth at 34°C. Both domains have to be mutated to inactivate the differentiating function. The inability of these mutant receptors to induce differentiation correlates with mitogen-activated protein kinase activation. In contrast, inhibitors of phosphatidylinositol 3-kinase have no effect on IGF-I- mediated differentiation of H19-7 cells, although they do inhibit the mitogenic response. INTRODUCTION The role of the IGF-IR 3 in cell growth, transformation, and protec- tion from apoptosis has been well established in our laboratory and several other laboratories (reviewed in Refs. 1 and 2). The IGF-IR also promotes differentiation in different cell types, such as myo- blasts, osteoblasts, hemopoietic cells (reviewed in Ref. 3), and macro- phages (4). There is also substantial evidence that the IGF-IR plays a role in neural differentiation, beginning with the previous observa- tions on oligodendrocytes (5) and neuronal cells, where the IGFs promoted neurite outgrowth and tubulin mRNA production (6 – 8). Additional references on the participation of the IGF-IR in neuronal differentiation can be found in two recent reviews by Leventhal et al. (9) and Anlar et al. (10). Even more interesting is a recent study by Arsenijevic and Weiss (11), in which the authors state that IGF-I is a differentiation factor for central nervous system stem cell-derived neuronal precursors. These findings should not be construed as mean- ing that the IGF-IR is the sole or even the most important receptor in neuronal differentiation. However, they indicate a role of the IGF-IR in this process, in association with other growth factor receptors. Given a role of the IGF-IR in neuronal differentiation, it is reason- able to ask how the IGF-IR participates in the differentiation of neuronal cells. One approach is to determine the domains of the IGF-IR required for neuronal differentiation because the identification of these domains could give important clues on the mechanisms involved. For this purpose, we infected H19-7 cells with retroviral vectors expressing a WT or several mutants of the human IGF-IR. H19-7 cells are rat hippocampal cells that have been conditionally immortalized by transducing a retroviral vector expressing a temper- ature-sensitive SV40 large T antigen (12). This cell line proliferates at the permissive temperature (34°C) in response to epidermal growth factor or serum and differentiates to a neuronal phenotype in N2 medium supplemented with bFGF at the nonpermissive temperature (39°C; Refs. 12 and 13). Differentiated H19-7 cells do not respond to serum, extend neurites, and express neuronal markers, such as neu- rofilament proteins and brain type II sodium channels, and display action potentials (12–14). Cells similarly immortalized by a temper- ature-sensitive SV40 large T antigen show region-specific neuronal differentiation on transplantation into rat brains (15, 16). In the present experiments, we wished first to establish whether the activated IGF-IR could induce differentiation of H19-7 cells at 39°C and then to determine the domains in the IGF-IR required for the induction of differentiation. As a control, we examined the WT and mutant IGF-IRs for their ability to respond to IGF-I with mitogenesis at 34°C. We show here that tyrosine 950 and the serines 1280 –1283 in the COOH terminus of the IGF-IR are necessary for differentiation of H19-7 cells but are dispensable for IGF-I-mediated mitogenesis. This finding clearly separates the mitogenicity of the IGF-IR from its ability to modulate differentiation in neuronal cells. We also carried out preliminary experiments on IGF-IR signaling in these cells at the two temperatures. The inability of certain mutant receptors to promote differentiation correlates with their inability to give a sustained acti- vation of ERK1/2, and differentiation is inhibited by MEK inhibitors, thus confirming previous results by other investigators on the role of MAPK in the differentiation of neuroblastoma cells (8, 17, 18). However, inhibitors of the PI3K pathway have no effect on the differentiation of H19-7/IGF-IR cells, although they completely in- hibit the mitogenic response at 34°C. MATERIALS AND METHODS Cell Lines. H19-7 are rat hippocampal cells immortalized by transduction of a retroviral vector expressing a temperature-sensitive SV40 large T antigen (12). H19-7 cells are maintained in DMEM supplemented with 10% fetal bovine serum and 200 g/ml G418 (selection for the T antigen plasmid) in flasks coated with 15 g/ml poly-L-lysine (Sigma). R12, R508, R600, and p6 cells are mouse embryo fibroblasts expressing different numbers of human IGF receptor as described previously (19, 20). Plasmids and Retroviral Vectors. pHIT60 and pHIT123 were kindly provided by Dr. Alan Kingsman (Oxford University, Oxford, United King- dom) and are described elsewhere (21). pHIT60 contains the murine leukemia virus gag/pol cassette under the control of the human cytomegalovirus imme- diate early promoter, whereas pHIT123 contains the human cytomegalovirus, i.e., driven murine leukemia virus ecotropic envelope. pMSCV vectors were kindly provided by Dr. Robert G. Hawley (University of Toronto, Toronto, Canada). The human IGF-IR receptor cDNA was excised from the CVN plasmid (20, 22) by HindIII and HpaI digestion, filled in with Klenow Received 11/17/99; accepted 2/17/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants CA 56309 and AG 16291 (to R. B.) and NS 33858 (to M. R. R.). 2 To whom requests for reprints should be addressed, at Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, 624 BSLB, Philadelphia, PA 19107. Phone: (215) 503-4507; Fax: (215) 923-0249; E-mail: r_baserga@lac.jci.tju.edu. 3 The abbreviations used are: IGF-IR, type I insulin-like growth factor receptor; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3'-kinase; IGF, insulin-like growth factor; bFGF, basic fibroblast growth factor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein/ERK kinase; SFM, serum-free medium; NF68, neurofilament protein 68; BrdUrd, 5-bromo-2'-deoxyuridine; WT, wild- type; FGF, fibroblast growth factor; IRS-1, IR substrate 1; IR, insulin receptor. 2263