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 neuroblastoma 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 caused cell detachment at concentrations >0.3 ,g/ml; in the Nb2a clone of mouse neuroblastoma cells (Fig. 1B). however, at 0.1 ,g/ml it did not block neurite extension After 4 hr in serum-free medium, -60% of the cells had (Table 1). Thus, the ability of thrombin to block differentia- neurites longer than one cell diameter, and many had several tion appeared to result from a specific effect rather than (up to seven) neurites. Many cells with branched neurites general proteolysis, which might simply detach neurites from were also observed. the culture dish. Consistent with this conclusion is the finding Addition ofhighly purified thrombin (0.1 ,ug/ml) at the time that thrombin (1 ,ug/ml) had no detectable effect on the the neuroblastoma cells were switched to serum-free medium attachment of neuroblastoma cells to the culture dishes. blocked neurite outgrowth; the percentage of differentiated Thrombin (1 ,g/ml) did not increase the rate of thymidine cells did not increase above the 10%o level that was charac- incorporation or cell number in serum-free cultures of neu- teristic of serum-containing cultures (Fig. 1C; Table 1).. roblastoma cells, indicating that the inhibition of neurite Thrombin blocked neurite extension at extremely low con- outgrowth was not due to an effect on cell proliferation. centrations (Fig. 2); a half-maximal response occurred at -2 Thrombin also did not affect cell viability, as judged by ng/ml (50 pM) and a maximal response was observed at 100 trypan blue dye exclusion. ng ofthrombin per ml. DIP-thrombin was 500-1000 times less Neurite Retraction by Thrombin. We also tested the hy- potent in preventing neurite outgrowth than active thrombin pothesis that thrombin might cause retraction of neurites in (Fig. 2). This preparation contained -0.1% residual thrombin neuroblastoma cells that had been induced to differentiate by activity (data not shown), indicating that the proteolytic A B C .7 ft a)

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9 ft .r v * + 10 100 *. Thrombin (ng/ml) f FIG. 2 Ability of thrombin to block neurite outgrowth and produce neurite retraction in serum-free cultures of neuroblastoma cells. Cells were changed from DMEM containing 10% fetal calf serum to serum-free DMEM containing the indicated concentrations FIG. 1 Photomicrographs of unfixed neuroblastoma cells. Cells of thrombin (o) or DIP-thrombin (A) for 5 hr prior to fixing cells and were grown for 2 days in DMEM containing 10%6 fetal calf serum and scoring for neurite outgrowth. In parallel cultures, cells were first then exposed to the following conditions: (A) 4 hr in DMEM incubated in serum-free DMEM for 2 hr to induce neurite outgrowth; containing 0.8% fetal calf serum; (B) 4 hr in serum-free DMEM; (C) the indicated concentrations of thrombin were then added for an 4 hr in serum-free DMEM containing thrombin (0.1 Mg/ml). (Bar = additional 3 hr prior to fixing the cells and scoring for neurite 20 ,m.) outgrowth (e). Downloaded by guest on September 24, 2021 3442 Cell Biology: Gurwitz and Cunningham. Proc. NatL Acad. Sci. USA 85 (1988)

80 per ml compared to 10 ng/ml for the same medium before it was added to cells. The increased thrombin (or thrombin-like protein), however, appeared not to be released from the cells *z 60 since incubation of the medium in the absence of cells for 3 days at 37°C also produced almost a 3-fold increase in i 40 thrombin (28 ng/ml). Similar measurements on medium

0) containing 10% fetal calf serum from other sources also 0 I~~~~~~~~~ 20 /~~~~~~~~f+/ revealed substantial increases in thrombin concentrations - -_ after incubation in the absence ofcells for 3 days at 37°C; the o initial thrombin concentrations were 3.4, 3.3., and 9.0 ng/ml, - O 2 4 6 while the final concentrations were 9.6, 28, and 35 ng/ml, Time (h) respectively. These latter concentrations of serum-derived FIG. 3 Time course of induction of neurite outgrowth by serum- thrombin would be sufficient to account for most, but not all, free medium and retraction of neurites by thrombin. At time zero, ofthe ability ofmedium containing 10%o serum to maintain the neuroblastoma cultures were switched from DMEM containing 10%o neuroblastoma cells in an undifferentiated state (Fig. 2). fetal calf serum to serum-free DMEM (o); at 3.5 hr (arrow) thrombin Finally, it should be pointed out that the serum-free medium (0.1 /Lg/ml) was added to some of these cultures (A). In a parallel set from 2-day cultures of differentiated neuroblastoma cells did of cultures, thrombin (0.1 pug/ml) was added when the cells were not contain significantly more thrombin than the same me- switched to serum-free medium at time zero (0); at 3.5 hr (arrow), dium not exposed to cells (Table 2). This measurement was some of these cultures were switched to serum-free medium con- made at 2 days because there was significant cell detachment taining no thrombin (A). by 3 days in serum-free medium. incubation in serum-free medium for 4 hr (Fig. 3). Addition The following experiments provided additional evidence of 0.1 ;Lg of thrombin per ml to these differentiated cells that thrombin in the serum-containing culture medium could brought about a rapid retraction ofneurites. Four hours later, regulate neuroblastoma differentiation and that this regula- the appearance ofthese cells was indistinguishable from cells tion was reversible. Addition of heparin (0.8 unit/ml) to the that had been exposed to the same concentration ofthrombin culture medium containing 10%o serum that was incubated at throughout the 4 hr period in serum-free medium-i.e., only 37°C in the presence of cells for 3 days reduced the thrombin 409o of the cells had extended neurites (Fig. 3). To further concentration from 27 ng/ml to 0.8 ng/ml (Table 2), presum- compare the ability of thrombin to bring about neurite ably by accelerating the inactivation of thrombin by anti- and block neurite thrombin III. When the same concentration of heparin was retraction to extension caused by serum- added to neuroblastoma cells in medium containing 0.8% free medium, we compared the serum doses required for both serum, it produced rapid neurite outgrowth in -80% of the effects. Although the required concentrations were very cells (Fig. 4). Importantly, this effect of heparin was blocked similar, slightly higher doses were required for neurite by simultaneous addition of thrombin (Fig. 4A). Thrombin retraction (Fig. 2). also brought about retraction of neurites in neuroblastoma Role of Serum-Derived Thrombin in Control of Neuroblas- cells previously treated with heparin (Fig. 4B). The neurite- toma Differentiation. The results described above suggested promoting effect of heparin was abolished when it was that the low level of neurites on neuroblastoma cells cultured pretreated with heparinase (data not shown). in the presence of serum might be due to thrombin that was Induction of Neurite Outgrowth by Protease Inhibitors Is either derived from serum or released by the cells. To test Blocked and Reversed by Thrombin. We next took advantage these possibilities, a specific and sensitive assay for thrombin of studies by Monard and colleagues, which showed that was used that we developed earlier (25). As described in added thrombin could block the neuroblastoma differentia- Materials and Methods, the assay is based on two different tion produced by serum-free medium conditioned by glioma highly specific thrombin recognition steps. Although the cells (15). They subsequently purified a glial-derived neurite- neurite outgrowth assays were conducted in medium containing 0.8% serum (14-16), the thrombin assays were conducted with medium containing 10% serum to permit more A B accurate measurements. Table 2 shows that medium containing 10% fetal calf serum that had been exposed to neuroblastoma cells for 3 days contained -27 ng ofthrombin ~60- Table 2. Thrombin levels detected in cell culture media Addition(s) Thrombin, ng/ml 420- DMEM not exposed to cells None 0.1 ± 0.2 10%o fetal calf serum (day 0) 10 ± 2.6 10% fetal calf serum (day 3) 28 ± 3.8 o 0.o0 o.1 10 -1 0 2 4 6 8 DMEM exposed to cells Thrombin (tig/ml) Time (h) None (day 2) 0.5 ± 0.2 10% fetal calf serum (day 3) 27 ± 1.7 FIG. 4 Induction of neurite outgrowth by heparin and its inhibi- 10%o fetal calf serum and heparin tion and reversal by thrombin. (A) The medium of neuroblastoma ± cells was changed from DMEM containing 10% fetal calf serum to (0.8 unit/ml) (day 3) 0.8 0.5 DMEM containing 0.8% fetal calf serum. Then, heparin (0.8 unit/ml) Media were incubated at 37°C in the presence or absence of cells and the indicated concentrations ofthrombin were added. After 4 hr, for the indicated times and then assayed for thrombin by using the cells were fixed and scored for neurite outgrowth. (B) The '25I-labeled hirudin. Samples of purified human thrombin were medium on neuroblastoma cells was changed to DMEM containing assayed in parallel to construct a standard curve, which for the 0.8% fetal calf serum at time zero and the percentage of cells with experiment shown had a slope of 8320 cpm per ng thrombin and a neurites was scored (o). In parallel cultures, heparin (0.8 unit/ml) was background of215 cpm that was subtracted from all readings. Values added at time zero (e); at 3.5 hr (arrow), thrombin (1 ug/ml) was are means of duplicate determinations ± 1 SEM. added to some of these cultures (A). Downloaded by guest on September 24, 2021 Cell Biology: Gurwitz and Cunningham. Proc. Natl. Acad. Sci. USA 85 (1988) 3443 promoting factor and showed it was a 43,000-Da inhibitor of Table 3. Effect of thrombin and urokinase on induction of thrombin and urokinase (16, 18). Recent studies have shown neurite outgrowth by protease inhibitors that the glial-derived neurite-promoting factor is identical to Addition (per ml) % differentiated cells PN-1 (32-34), an inhibitor of thrombin, and urokinase, Experiment 1 originally identified in serum-free culture medium from Control 14 ± 4 human fibroblasts (35). In view of these results, it was PN-1 (2,ug) 55 ± 5 important to examine the stoichiometry by which thrombin ± 2 of the PN-1 (2 dig) + thrombin (3 jug) 11 blocks the neurite outgrowth-promoting activity glial- PN-1 (2 j.g) + DIP-thrombin (3 Aug) 57 ± 3 derived neurite-promoting factor/PN-1 and to determine PN-1 (2 ug) + urokinase (3 ug) 16 ± 3 whether thrombin could reverse the differentiation it pro- Experiment 2 duced. Control 10 ± 2 Fig. 5A shows that purified PN-1 induced neurite out- Hirudin (1 unit) 80 ± 3 growth in the neuroblastoma cells and that a half-maximal Hirudin (1 unit) + thrombin (0.3 ,ug) 45 ± 4 response occurred at -0.7 ,g/ml. Added thrombin blocked 81 ± 4 this effect of PN-1 at 2:1 stoichiometric concentrations (Fig. Hirudin (1 unit) + urokinase (3 ,ug) 5), consistent with the finding that 50% of the PN-1 was Medium on neuroblastoma cultures was changed to DMEM capable of forming complexes with thrombin (data not containing 0.8% fetal calf serum together with the tested proteins. shown). The blockage by thrombin required its proteolytic After 4 hr, the cells were fixed and scored for neurite outgrowth. activity, since DIP-thrombin did not prevent neurite out- Values are means of duplicate determinations ± 1 SEM. growth by PN-1 (Table 3). Finally, it is noteworthy that added thrombin and 50% in the presence of thrombin (1 ,g/ml). thrombin produced neurite retraction in neuroblastoma cul- Thus, the ability of thrombin to block neurite outgrowth tures induced to differentiate by added PN-1 (Fig. SB). and Similar results were obtained in experiments that used depended on the agent used to induce differentiation hirudin, a highly specific inhibitor ofthrombin (Fig. 6). These appeared to results from specific rather than nonspecific studies showed that hirudin induced neurite outgrowth, but effects of proteolysis. this was blocked by stoichiometric concentrations of thrombin, and that thrombin reversed the neuroblastoma differen- DISCUSSION tiation produced by hirudin. As expected, urokinase did not block the neurite outgrowth activity of hirudin even at a The experiments reported here address two previously de- 10-fold higher concentration than required for thrombin to scribed but poorly understood ways to induce neuroblastoma half maximally block the hirudin effect (Table 3). Together, differentiation. The first is to switch cells to serum-free the results in this section showed that the modulation of medium. This induces neurite outgrowth not only in neuro- neurite outgrowth by thrombin did not depend on the par- blastoma cells (5-9) but also in several primary cultures of ticular protease inhibitor used to stimulate neurite outgrowth. central and peripheral neurons (27-31). The second is to add Induction ofNeurite Outgrowth by Bt2cAMP or PGE1 Is Not certain inhibitors of serine proteases (15, 16), particularly the Blocked by Thrombin. Agents that elevate intracellular cAMP glial-derived neurite-promoting factor, which inhibits throm- levels, such as Bt2cAMP and PGE1, are known to induce bin and urokinase (18) and stimulates neurite outgrowth in neuroblastoma differentiation (1-4). To determine whether cultured neuroblastoma cells (15, 16) and primary neurons (17). thrombin blocked their effects, various concentrations of The present studies imply a role of thrombin in both of the thrombin were added simultaneously with 1 mM Bt2cAMP or methods described above to induce neurite outgrowth from 50 ,uM PGE1 to neuroblastoma cultures in medium containing neuroblastoma cells. Its role in the regulation by serum is 0.8% serum. These were the minimal concentrations of each suggested by findings that: (i) the neurite outgrowth that is agent required to produce neurite outgrowth in >50% of the induced by removal of serum was prevented by thrombin cells. Thrombin was only marginally effective in blocking addition, (ih) added thrombin produced neurite retraction in neurite outgrowth induced by Bt2cAMP and PGE1; at 5 hr the neuroblastoma cells that had been induced to differentiate by percentage of cells with neurites was =60% in the absence of IA Bt BI ,,80 a) ,60_ Z60 -"'' =a 40 .3: 40 is 3C40-

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I I 0 10 100 -1 0 2 4 6 nM Time (h) 0 0.010.1 10 -1 0 2 4 6 8 Thrombin (1ig/ml) Time (h) FIG. 5 Induction of neurite outgrowth by PN-1 and its inhibition and reversal by thrombin. (A) The medium on neuroblastoma cells FIG. 6 Induction of neurite outgrowth by hirudin and its inhibi- was changed from DMEM containing 10%o fetal calf serum to DMEM tion and reversal by thrombin. (A) The medium on neuroblastoma containing 0.8% fetal calf serum. Then, the indicated concentrations cells was changed from DMEM containing 10o fetal calf serum to of PN-1 (o) or 46 nM PN-1 (2 p.g/ml) along with the indicated DMEM containing 0.8% fetal calf serum. Then, hirudin (1 unit/ml) concentrations of thrombin (o) were added. After 4 hr, the cells were and the indicated concentrations ofthrombin were added. After 4 hr, fixed and scored for neurite outgrowth. (B) The medium on neuro- the cells were fixed and scored for neurite outgrowth. (B) The blastoma cells was changed to DMEM containing 0.8% fetal calf medium on neuroblastoma cells was changed to DMEM containing serum at time zero, and the percentage of cells with neurites was 0.8% fetal calf serum at time zero, and the percentage of cells with scored in cultures without additions (o). In parallel cultures, PN-1 (2 neurites was scored (o). In parallel cultures, hirudin (1 unit/ml) was ,ug/ml) was added at time zero (o); at 3.5 hr (arrow), thrombin (2 added at time zero (o); at 3.5 hr (arrow), thrombin (1 ug/ml) was ,ug/ml) was added to some of these cultures (A). added to some of these cultures (A). Downloaded by guest on September 24, 2021 3444 Cell Biology: Gurwitz and Cunningham. Proc. Natl. Acad Sci. USA 85 (1988) serum removal, (iiR) assays for thrombin in serum-containing 2. Prasad, K. N. & Hsie, A. W. (1971) Nature (London) New medium that inhibited neurite outgrowth indicated that much Biol. 233, 141-142. but not all ofthe inhibition could be accounted for by thrombin 3. Prasad, K. N., Sahu, S. K. & Sinha, P. K. (1976) J. Natl. Cancer Inst. 57, 619-629. that was produced during the incubation of this medium at 4. Prasad, K. N. (1972) Nature (London) New Biol. 236, 49-51. 370C, and (iv) heparin addition not only reduced thrombin 5. Bottenstein, J. E. & Sato, G. H. (1979) Proc. Natl. Acad. Sci. levels in serum-containing medium but also stimulated neurite USA 76, 514-517. outgrowth. 6. Schubert, D., Humphreys, S., Baroni, C. & Cohn, M. (1969) The conclusion that thrombin plays a primary role in the Proc. Natl. Acad. Sci. USA 64, 316-323. of neuroblastoma differentiation by protease inhib- 7. Schubert, D., Humphreys, S., De Vitry, F. & Jacob, F. (1971) regulation Dev. Biol. 25, 514-546. itors is based on findings that two quite different thrombin 8. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., inhibitors, PN-1 (identical to the glial-derived neurite- Kimes, B., Patrick, J., Steinbach, J. H., Culp, W. & Brandt, promoting factor) and hirudin, indistinguishably induced neu- B. L. (1974) Nature (London) 249, 224-227. rite outgrowth. Moreover, thrombin similarly blocked the 9. Seeds, N. W., Gilman, A. G., Amano, T. & Nirenberg, M. W. neurite outgrowth activity of both PN-1 and hirudin and (1970) Proc. Nati. Acad. Sci. USA 66, 160-167. reversed their effects by bringing about neurite retraction in 10. Marchisio, P. C., Osborn, M. & Weber, K. (1978) J. Neurocy- their continued presence. Although the neurite-inducing ac- tol. 7, 571-582. 11. Olmsted, J. B. (1981) J. Cell Biol. 89, 418-423. tivity of PN-1 could also be blocked by urokinase (Table 3), 12. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. Natl. this was undoubtedly due to the ability ofthis protease to form Acad. Sci. USA 69, 258-263. complexes with PN-1 (35) and thus prevent it from inactivating 13. Augusti-Tocco, G. & Sato, G. (1969) Proc. Natl. Acad. USA thrombin in the medium. Consistent with this conclusion is the 64, 311-315. finding that after switching neuroblastoma cells to serum-free 14. Schurch-Rathgeb, Y. & Monard, D. (1978) Nature (London) medium, urokinase, unlike thrombin, did not block neurite 273, 308-309. outgrowth and did not produce neurite retraction. Together, 15. Monard, D., Niday, E., Limat, A. & Solomon, F. (1983) Prog. Brain Res. 58, 359-364. these results indicate that the induction of neurite outgrowth 16. Guenther, J., Nick, H. & Monard, D. (1985) EBMO J. 4, by PN-1 and hirudin results from inhibition of thrombin. 1963-1966. Several other observations imply that thrombin might 17. Monard, D., Gloor, S., Sommer, J. & Nick, H. (1987) J. participate in the intricate regulation of neuronal differenti- Neurochem. 48, Suppl. 517 (abstr.). ation in vivo. First, the glial-derived neurite-promoting factor 18. Stone, S. R., Nick, H., Hofsteenge, J. & Monard, D. (1987) identified by Monard and colleagues undoubtedly functions Arch. Biochem. Biophys. 252, 237-244. in the brain since it is released both by glioma (16) and 19. Krystosek, A. & Seeks, N. W. (1981) Science 213, 1532-1534. 20. Krystosek, A. & Seeds, N. W. (1981) Proc. Natl. Acad. Sci. primary astrocyte (14, 34) cultures and its mRNA can be USA 78, 7810-7814. detected in rat brain (32). Although this inhibitor inactivates 21. Soreq, H., Miskin, R., Zutra, A. & Littauer, U. Z. (1983) Dev. both thrombin and urokinase (18), the evidence presented Brain Res. 7, 257-269. earlier and here strongly indicates that its neurite-inducing 22. Snider, R. M., McKinney, M., Fenton, J. W. & Richelson, E. activity depends on its ability to inhibit thrombin. Second, (1984) J. Biol. Chem. 259, 9078-9081. specific binding sites for thrombin, with a Kd of =1 x 10-9 23. Snider, R. M., McKinney, M. & Richelson, E. (1986) Semin. M, have been detected on mouse neuroblastoma cells (clone Thromb. Hemostasis 12, 253-262. NlE-115) (23); it has been suggested that these sites mediate 24. Snider, R. M. (1986) Ann. N. Y. Acad. Sci. 485, 310-313. 25. Low, D. A. & Cunningham, D. D. (1982) J. Biol. Chem. 257, the ability of thrombin to increase intracellular cGMP levels 850-858. (22-24). Whatever their role, it seems likely that cells that 26. Farrell, D. H., Van Nostrand, W. E. & Cunningham, D. D. possess them potentially interact with thrombin either after (1986) Biochem. J. 237, 907-912. injury or during normal regulatory events. It is noteworthy 27. Adler, R. (1982) J. Neurosci. Res. 8, 165-177. that binding ofthrombin has also been detected in primary rat 28. Davis, G. E., Skaper, S. D., Manthorpe, M., Moonen, G. & brain cultures (36) and in homogenates of human brain and Varon, S. (1984) J. Neurosci. Res. 12, 29-39. spinal cord (37). Finally, preliminary studies indicate that rat 29. Luduena, M. A. (1973) Dev. Biol. 33, 470-476. brain contains mRNA for prothrombin (S. Wagner, A. Lau, 30. Ziller, C. & Le Douarin, N. M. (1983) Birth Defects Orig. Artic. P. Isackson, and D.D.C., unpublished results). In view of Ser. 19, 251-261. a 31. Ziller, C., Dupin, E., Brazeau, P., Pauline, D. & Le Douarin, these findings, the possible involvement of thrombin (or N. M. (1983) Cell 32, 627-638. thrombin-like protease) in the delicate balance between 32. Gloor, S., Odink, K., Guenther, J., Nick, H. & Monard, D. stimulatory and inhibitory signals that govern neurite out- (1986) Cell 47, 687-693. growth in vivo should be considered in future studies. 33. McGrogan, M., Gohari, J., Li, M., Hsu, C., Scott, R., Simon- sen, C. & Baker, J. B. (1988) BiolTechnology 6, 172-177. We thank Drs. Steven L. Wagner and William E. Van Nostrand for 34. Rosenblatt, D. E., Cotman, C. W., Nieto-Sapedro, M., Rowe, helpful discussions, Ramin Davidofffor technical assistance, and Dr. J. W. & Knauer, D. J. (1987) Brain Res. 415, 40-48. John W. Fenton II for thrombin. This work was supported by 35. Baker, J. B., Low, D. A., Simmer, R. L. & Cunningham, National Institutes ofHealth Grant CA12306. D.G. was supported by D. D. (1980) Cell 21, 37-45. a Chaim Weizmann Postdoctoral Fellowship. 36. Means, E. D. & Anderson, D. K. (1986) Ann. N. Y. Acad. Sci. 485, 314-322. 1. Furmanski, P., Silverman, D. P. & Lubin, M. (1971) Nature 37. McKinney, M., Snider, R. M. & Richelson, E. (1983) Mayo (London) 233, 413-415. Clin. Proc. 58, 829-831. Downloaded by guest on September 24, 2021