Thrombin Modulates and Reverses Neuroblastoma Neurite Outgrowth (Protease Nexin-1/Glial-Derived Neurite Promoting Factor/Heparin/Hirudin) DAVID GURWITZ and DENNIS D
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Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3440-3444, May 1988 Cell Biology Thrombin modulates and reverses neuroblastoma neurite outgrowth (protease nexin-1/glial-derived neurite promoting factor/heparin/hirudin) DAVID GURWITZ AND DENNIS D. CUNNINGHAM* Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92717 Communicated by John M. Buchanan, January 19, 1988 ABSTRACT Previous studies have shown that neuroblas- toma cells (14-16) and primary neurons (17). This glial- toma cells and several types ofprimary neuronal cells in culture derived neurite-promoting factor inactivates two protein- rapidly extend neurites when switched from serum-containing ases, thrombin and urokinase (18), that have been implicated to serum-free medium. The present studies on cloned neuro- in the control of neuronal/neuroblastoma cell differentiation. blastoma cells show that thrombin blocked this spontaneous Studies on urokinase have shown that it is localized to the differentiation at 2 nM with a half-maximal potency of 50 pM. neuronal growth cone (19) and have indicated that it is This required the catalytic activity of thrombin and was involved in neuronal migration (20, 21). Studies on thrombin reversed upon thrombin removal. Thrombin also caused cells have shown that it stimulates cGMP formation in neuroblas- in serum-free medium to retract their neurites at equally low toma cells and that these cells possess specific binding sites concentrations. Two other serine proteases, urokinase and for thrombin (22-24). Effects ofthrombin beyond this second plasmin, did not block or reverse neurite extension even at messenger, however, have not yet been identified in these 100-fold higher concentrations. A specific assay for thrombin cells. Hirudin, a specific inhibitor of thrombin, stimulates indicated that thrombin detected in serum-containing medium neurite outgrowth from neuroblastoma cells (15), suggesting from neuroblastoma cultures was derived from serum and that the possibility that thrombin might be involved in the control it was likely responsible for much of the known capacity of of neuronal differentiation. serum to maintain neuroblastoma cells in a nondifferentiated In view of these considerations, we evaluated the hypoth- state. This was supported by the rinding that heparin addition esis that thrombin might induce neurite retraction and mod- reduced the thrombin concentration in serum-containing me- ulate neurite outgrowth by certain agents. Cloned neuroblas- dium and stimulated neurite outgrowth from neuroblastoma toma cells were used in these studies to determine whether cells in serum-containing medium. Studies on the ability of thrombin had direct effects on neurite outgrowth in the thrombin to modulate neurite outgrowth by other agents absence of other cell types. showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hiru- MATERIALS AND METHODS din. Thrombin, however, did not block the neurite-promoting Materials. Purified human a-thrombin (2750 NIH units/mg) activity of dibutyryl cAMP or prostaglandin El. These results was generously supplied by John W. Fenton II (New York suggest a specific role for thrombin in control of neurite State Department of Health, Albany). Thrombin, whose outgrowth. catalytic site serine residue was derivatized with a diiso- propylphospho group (DIP-thrombin), was prepared as de- Cloned murine neuroblastoma cells have been widely used as scribed (25). Protease nexin-1 (PN-1) was purified from a model system for neuronal cells since they can be induced serum-free medium conditioned by normal human foreskin to differentiate in culture by stimuli such as dibutyryl cAMP fibroblasts cultured on microcarrier beads as described (26). (Bt2cAMP) (1-3), prostaglandin E1 (PGE1) (4), and serum Human urokinase was from Calbiochem-Behring (catalogue removal (5-9). Differentiation is observed morphologically as no. 672081) and human plasmin was from Boehringer Mann- neurite extension, a process that requires assembly of mi- heim (catalogue no. 602361). Bovine trypsin (type XIII), crotubules (10) and is blocked by microtubule-disrupting hirudin (2000 units/mg), heparin (168 units/mg), Bt2cAMP, agents (7, 11). Neurite outgrowth is paralleled by increased PGE1, and protein A-Sepharose were purchased from Sigma. activities of several enzymes related to neural function such Cell Culture. Mouse neuroblastoma subclone Nb2a was as tyrosine hydroxylase, choline acetyltransferase, and ace- purchased from the American Type Culture Collection. The tylcholine esterase (12, 13). Thus, despite some abnormalities cells were cultured in 100-mm tissue culture dishes (Coming) due to their neoplastic character, cloned neuroblastoma cells in Dulbecco's modified Eagle's medium (DMEM) (Flow display a differentiation pattern similar to neural crest cells in Laboratories) containing 10% fetal calf serum (GIBCO), the developing brain. penicillin G (100 units/ml), and streptomycin (100 ,ug/ml). The Although much effort has been devoted to studies on cells were maintained at 37°C in a humidified atmosphere of differentiation of neuroblastoma cells, few studies have dealt 10% C02/90% air. Cells were subcultured at a 1:10 ratio every with physiological agents that can modulate or reverse this 3-4 days by trituration in cell culture medium (no trypsini- process. It is important to elucidate mechanisms of neurite zation is required with this cell line). retraction and inhibition of neurite outgrowth since guidance Neurite Outgrowth Assay. Cells (18,000 in a volume of 1 ml and elongation of axons in vivo are likely governed by an of DMEM containing 10% fetal calf serum) were plated in intricate interplay of positive and negative signals. With this 12-well (diameter 22 mm) cluster tissue culture dishes in mind, there is special interest in the work of Monard and (Corning). Two days later, the medium was replaced with 1 colleagues, who identified a glial-derived protease inhibitor ml of DMEM containing either 0.8% fetal calf serum (14-16) that stimulates neurite outgrowth from cultured neuroblas- Abbreviations: Bt2cAMP, dibutyryl cAMP; DIP-thrombin, diiso- The publication costs of this article were defrayed in part by page charge propylphosphothrombin; PGE1, prostaglandin E1; PN-1, protease payment. This article must therefore be hereby marked "advertisement" nexin-1. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3440 Downloaded by guest on September 24, 2021 Cell Biology: Gurwitz and Cunningham. Proc. Nati. Acad. Sci. USA 85 (1988) 3441 or no serum. Additions of test substances were made imme- Table 1. Modulation by proteases of neurite outgrowth in diately after the medium change, unless otherwise noted, serum-free medium using stocks that were at least 100 times the final specified Addition (per ml) % differentiated cells concentration. After incubation at 37°C for the indicated times, the medium was removed, and cells were immediately Control 57 + 4 fixed with 3% paraformaldehyde in phosphate-buffered sa- Thrombin (0.1 Mg) 12 ± 2 line. Cells were examined for neurite outgrowth by phase- Thrombin (1 ug) 12 ± 3 contrast microscopy; cells at DIP-thrombin (1 Ag) 41 ± 2 exhibiting least one clearly Urokinase (10 Ig) 49 ± 5 defined neurite equal to or longer than one cell diameter were Plasmin (10 Ag) 58 ± 4 scored as positive. At least 200 cells were scored in each well Trypsin (0.1 Mg) 60 ± 3 to determine the percentage of cells with neurites; assays were routinely carried out in duplicate wells. The medium on neuroblastoma cultures was changed to serum-free DMEM together with the tested proteins. After 16 hr, the cells were Hirudin Assay for Thrombin. This assay was carried out as fixed and scored for neurite outgrowth as described. The values described (25). The assay depends on the binding of 12511 shown are means of duplicate determinations ± 1 SEM. labeled hirudin to thrombin; the complexes are then precip- itated by using IgG from rabbits immunized with DIP- activity of thrombin was necessary for its effects on neuro- thrombin followed by protein A-Sepharose. Radioactivity blastoma cell differentiation. was measured in a y-counter. The specific activity of the The ability of thrombin to block spontaneous differentia- 1251-labeled hirudin was 47,500 cpm/ng. Rabbit anti-DIP- tion was rapidly reversible upon replacement of the throm- thrombin antiserum was prepared as described (25), purified bin-containing serum-free medium with serum-free medium on protein A-Sepharose, and used at a concentration of 16 lacking thrombin (Fig. 3). When thrombin was removed, ,g/ml. neurite extension rapidly occurred with a rate similar to the rate observed when neuroblastoma cells in serum-containing RESULTS medium were switched to serum-free medium. The maximal proportion of cells with extended neurites occurred within Thrombin Blocks Neurite Outgrowth Induced by Serum 2-3 hr after removal of thrombin. Removal. Neuroblastoma cells and several types of primary Two other serine proteases, urokinase and plasmin, were neuronal cells in culture rapidly differentiate upon removal of ineffective in blocking neurite extension even at 10 Ag/ml serum from the culture medium judged by neurite outgrowth (Table 1). Trypsin could not be used at high concentrations (5-9, 27-31). Removal of serum also led to neurite extension since it