Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2562-2566, April 1984 Neurobiology Insulin and insulin-like growth factor II permit nerve growth factor binding and the neurite formation response in cultured human neuroblastoma cells (axons/differentiation/nerve growth factor receptor/pheochromocytoma PC12) ESPERANZA RECIO-PINTO, FREDERICK F. LANG, AND DOUGLAS N. ISHII* Department of Pharmacology and Cancer Research Center, College of Physicians and Surgeons of Columbia University, New York, NY 10032 Communicated by I. S. Edelman, January 3, 1984 ABSTRACT In serum-free medium, SH-SY5Y human mouse saliva (16); purity was confirmed by the single band in neuroblastoma cells specifically and reversibly lost the capaci- polyacrylamide gels subjected to isoelectric focusing (pH ty to bind 1251-labeled nerve growth factor (NGF) to the high- 3.5-10), or NaDodSO4 gel electrophoresis and by bioassay affinity sites (slow sites) and to respond by neurite outgrowth, (17). Anti-insulin antiserum was from Cappel Laboratories unless physiological concentrations of insulin or insulin-like (Cochranville, PA). The cloned cell line SH-SY5Y (7) was a growth factor II were present. In serum-containing medium, kind gift from June L. Biedler and Barbara A. Spengler. anti-insulin antiserum decreased the neurite formation re- Cells between passage numbers 9 and 29 were studied. The sponse to NGF, and insulin supplementation increased the cloned PC12 cell line (18) was the kind gift of Lloyd A. number of available NGF slow sites. The low-affinity NGF fast Greene and was subcloned prior to use. sites are absent from SH-SY5Y cells and did not emerge on Cell Culture. Cells were maintained in the logarithmic treatment with insulin. Insulin potentiated the induction of phase of growth in Roswell Park Memorial Institute medium neurites by NGF in rat pheochromocytoma PC12 cells also. 1640 (RPMI 1640) supplemented with 12% fetal calf serum, These results implicate a wider role for insulin and its homo- sodium penicillin G at 50 units/ml, and streptomycin sulfate logs in the nervous system. at 25 ,ug/ml at 370C in humidified 5% C02/95% air (4, 5). Neurite Outgrowth. For studies in serum-containing medi- Nerve growth factor (NGF) is important to the development um (SCM), cells were seeded onto microwell plates in the of the sympathetic and sensory neurons of vertebrates and above growth medium and permitted to attach for 3 days, can cause outgrowth of axons in vitro and in vivo (1-3). We and then fresh warm RPMI 1640 medium with 12% fetal calf have used the human neuroblastoma SH-SY5Y cell as a serum and various test solutions was added. For studies in model in which to study the mechanism of neurite formation SFM, after attachment, cells were washed three times with (4-6). Cloned SH-SY5Y cells (7) respond to NGF with in- warm RPMI 1640 medium, then warm RPMI 1640 medium creased neurite outgrowth (4, 8) and veratridine-dependent containing various test solutions was added. The percentage Na' uptake (8). The neurites show ultrastructural maturation of cells bearing neurites was determined by replicate counts in cells treated with NGF (8) and end in typical neuronal on more than 100 cells in several randomly chosen fields un- growth cones (9). der low-power modulation-contrast microscopy as described High-affinity sites (slow sites) and low-affinity sites (fast (4, 5). sites) that bind 1251-labeled NGF are present in sensory (10, Binding Assay. Purified NGF was iodinated (0.1-0.2 mol 11), sympathetic (12), and pheochromocytoma PC12 (13, 14) of 125i per mol of NGF) with lactoperoxidase (4). Cells were cells. Both sites are also present in some human neuroblasto- detached from nonconfluent monolayer cultures in Hanks' ma cell lines, but only the slow type site is present in SH- salts solution with 1 mM EDTA. Cells (2-4 x 106 per ml) SYSY cells (4), suggesting that binding to the fast sites is not were resuspended in RPMI 1640 medium containing bovine required for neurite outgrowth. The hypothesis that the slow serum albumin at 5 mg/ml and incubated for 1 hr at 37°C. In sites are the receptors for neurite outgrowth would be con- the absence of this incubation, down modulation of binding siderably more attractive if it were possible to activate and can often, but not always, be observed (4). Then, 0.1 nM (2.5 inactivate the capacity of these sites to bind NGF and to ng/ml) 125I-labeled NGF was added. Other conditions are show corresponding activation and inactivation of the capac- described in the legends. In cell lines without fast sites, non- ity to respond by neurite outgrowth. specific binding was assayed in parallel incubations addition We show that SH-SY5Y cells reversibly lose virtually all ally containing 100-fold excess NGF at the onset, and this capacity to bind NGF and respond by neurite outgrowth in binding was subtracted from other values. However, a 1000- serum-free media (SFM). Moreover, both capacities are re- fold excess of NGF is required to displace all specific bind tained in media containing insulin or insulin-like growth fac- ing in cell lines with lower affinity fast sites. Binding to fast tor II (IGF-II). sites is lost and binding to slow sites is quantitatively re tained after 10 min on ice with 1000-fold excess NGF, as in MATERIALS AND METHODS PC12 cells (13). Samples were centrifuged in needlenosed Materials. Pork pancreatic insulin (24 units/mg) and phor- 400-,ul capacity tubes (W. Sarstedt, Princeton, NJ) for 30 bol 12,13-dibutyrate (PBt2) were from Sigma. Insulin was sec. Tubes were quickly frozen in an acetone/dry ice bath, dissolved in 0.01 M HCl and stored at -20°C. Rat liver cell the tips were severed (tip volume was about 2 ,ul), and radio multiplication-stimulating activity, which is the same as activities were measured (4). IGF-II (15), of about 85% purity, was from Collaborative Re- Statistics. Values are means and SEM; n values are given search. The ,3 subunit of NGF was prepared from male Abbreviations: IGF-II, insulin-like growth factor II (rat liver multi- The publication costs of this article were defrayed in part by page charge plication-stimulating activity); NGF, nerve growth factor; PBt2, payment. This article must therefore be hereby marked "advertisement" phorbol 12,13-dibutyrate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2562 Downloaded by guest on September 27, 2021 Neurobiology: Recio-Pinto et aL Proc. Natl. Acad. Sci. USA 81 (1984) 2563 in legends. Analysis of variance and the Bonferroni multiple NGF plus 0.1 nM IGF-II, 51.9 ± 5.5%. The values are means t test (19) were used to compare experimental groups. Data ± range (n = 2 replicate cultures in which 400 cells were expressed as percentages were transformed to arc sin values scored). IGF-II alone directly enhanced neurite outgrowth; a prior to statistical analysis. suboptimal concentration for direct enhancement of neurite formation was studied. NGF alone had no significant activi- RESULTS ty even at a very high concentration. When NGF and IGF-II Effect of Insulin on SH-SY5Y Cell Neurite Formation in were added in combination, the neurite outgrowth response SFM. To avoid the variable amounts of serum components to NGF was potentiated. Thus, IGF-II, like insulin, had per- that may affect neurite formation, some tests were conduct- missive as well as direct effects on neurite outgrowth. ed in SFM, in which SH-SY5Y cells can survive at least a Effect of Anti-Insulin Antiserum on the SH-SY5Y Cell week without loss in number (4). Growth resumes on the Neurite Formation Response to Insulin, NGF, and PBt2. reintroduction of serum. The maximum neurite outgrowth Neurite outgrowth was increased in insulin-supplemented response to insulin is reached in 3 days in SFM. SCM (Table 1). Anti-insulin antiserum completely blocked In SFM, up to 0.75 ,uM NGF alone did not enhance neurite this increase. NGF increased the proportion of cells with outgrowth (Fig. 1A), showing that other serum factors are neurites from 27% to 70%. The anti-insulin antiserum, when required for the response. With 1 nM insulin alone, neurite added together with NGF, inhibited 77% of this increase, outgrowth was increased to about 13%. This will be referred showing that the low level of insulin normally present in se- to as the "direct" effect, since other serum factors were not rum was required for expression of a major fraction of the required. When 1 nM insulin was present, the simultaneous NGF activity. The anti-insulin antiserum also decreased the addition of various concentrations of NGF caused a syner- spontaneous neurite outgrowth to less than half the level in gistic, dose-dependent increase in neurite outgrowth. This untreated cultures; the residual spontaneous neurite out- shall be referred to as the "permissive" effect of insulin. The growth may be due to the activity of other serum factors. true sensitivity to NGF is obscured in Fig. LA because of its The antiserum does not, however, nonspecifically inhibit strong tendency to adsorb to culture vessel walls (20). The neurite outgrowth. Tumor promoters can reversibly increase inclusion of bovine serum albumin to reduce nonspecific ad- neurite formation in SH-SY5Y cells (5, 24). The promoter sorption resulted in a sharp shift in the NGF response curve PBt2 increased neurite outgrowth in a manner that was not to the left, such that the half-maximal response was at about inhibited by the anti-insulin antiserum (Table 1). 0.2-0.4 nM (Fig. 1B). The small increase in response to NGF These results suggest that the capacity of SH-SY5Y cells seen even in cultures untreated with insulin was probably to respond to NGF depends on the activity of serum insulin due to the activity of impurities in the albumin preparation.