JOURNAL OF CELLULAR PHYSIOLOGY 142155-162 (1990)
Neurite Outgrowth Activity of Protease Nexin-I on Neuroblastoma Cells Requires Thrombin Inhibition DAVID GURWITZ AND DENNIS D. CUNNINGHAM' Department of Microbiology and Molecular Cenetirs, College of Medicine, University of California, Irvine, CA 92717 Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1 :thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-I, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-I did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in (he presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin Ill, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin I l l and PN-I corresponded to their inhibition of thrombin. Together, [hew observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.
There is much evidence that several protease inhibitors can stimulate neurite outgrowth from certain cultured neuroblastoma cells and primary neurons (Monard et al., 1983; Guenther et al., 1985; Hawkins and Seeds, 1986; Pittman and Patterson, 1987; Gurwitz and Cunningham, 1988; Zurn et al., 1988; Hawkins and Seeds, 1989). This has led to the suggestion that a balance between proteases and protease inhibitors may be critical for the regulation of neurite outgrowth (Monard, 1987, 1988; Pittman and Patterson, 1987). Two proteases that have been implicated in this control are plasminogen activator and thrombin. Plasminogen activator is released from the growth cones of cultured neurons and could facilitate neurite outgrowth (Krystosek and Seeds, 1981). It has been suggested that thrombin could play an important role in this regulation since several thrombin inhibitors stimulate outgrowth (Monard et al., 1983) and since thrombin itself retracts neurites (Gurwitz and Cunningham, 1988). A protease inhibitor that could play a physiological role in the regulation of neurite outgrowth is the glial-derived neurite-promoting factor or glial-derived nexin. Previous studies showed that mouse neuroblastoma cells and chick sympathetic neurons extend neurites in response to this factor (Schurch-Ratgeb and Monard, 1978; Monard et al., 1983; Zurn et al., 1988; Gurwitz and Cunningham, 1988). This factor was re1990 WILEY-LISS, INC.
cently shown to be identical with protease nexin-1 (PN1)(Gloor et al., 1986; McGrogan et al., 19881, which is secreted by a variety of cultured cells and rapidly inhibits thrombin, urokinase, and plasmin by forming complexes with their catalytic site serine residues (Baker et al., 1980; Scott et al., 1985). The complexes bind back to the cells and are rapidly internalized and degraded (Low et al., 1981). It has been suggested that the neurite outgrowth activity of the glial-derived nexiniPN-1 depends on inhibition of thrombin since hirudin, another thrombin inhibitor, also stimulates neurite outgrowth in neuroblastoma cells (Monard et al., 1983; Gurwitz and Cunningham, 1988). Thrombin blocks the neurite outgrowth activity of the glial-derived nexin/PN-1 and also brings about retraction of neurites (Gurwitz and Cunningham, 1988). Urokinase also blocks the neurite outgrowth activity of the glialderived nexin/PN-1, although this could be due t o its ability to form complexes with PN-1 and thus reduce the concentration of free PN-1. Urokinase, unlike thrombin, does not bring about retraction of neurites (Gurwitz and Cunningham, 1988).
Received June 19, 1989; accepted September 14, 1989. *To whom reprint requestsicorrespondence should be addressed.
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Mannheim, Indianapolis). Porcine heparin (168 Uimg), bovine serum albumin (BSA, RIA grade), and protein A-Sepharose were from Sigma Chemical Go. (St. Louis). Na1251was from New England Nuclear (Boston). It was used to radioiodinate hirudin and thrombin using Iodogen (Pierce, Rockford, IL) (Baker e t al., 1980). Electrophoresis reagents and molecular weight markers were from Bio-Rad (Richmond, CA).
Preparation of pure PN-1:thrombin complexes Thrombin (1.4 pm) was incubated with 7 pm PN-1 for 2 h r a t 23°C. The unreacted PN-1 was removed by incubating the mixture with protein A-Sepharose coupled to a monoclonal antibody, which binds free but not 0 2 4 6 complexed PN-1 (Wagner et al., 1988). After incubation for 4 h r a t room temperature with shaking, the time ( h l mixture was centrifuged at 8,000g for 15 min, and the Fig. 2. Effect of PN-1 on neurite outgrowth in the absence of added complexes were recovered in the supernatant. The puthrombin; effect of subsequent. thrombin addition. The medium on rity of the complexes was verified by SDS polyacrylaneuroblastoma cells was changed to serum-free DME medium with mide gel electrophoresis (Laemmli, 1970). PN-1:urokieither no addition (01, 4 nM PN-1 (*),or 0.8 nM thrombin (A,. At the nase complexes were prepared using a similar protocol. times indicated by the arrows, 8 nM thrombin was added to the first two sets of cultures, while 4 nM PN-1 was added to the third set. Cells were scored for neurite outgrowth at the indicated time points.
Although the above studies indicate a role for thrombin in the stimulation of neurite outgrowth by the glial-derived nexiniPN-1, there are important unanswered questions regarding the molecules that interact with cells to produce this response. For example, it was recently suggested that complexes between the glialderived nexiniPN-1 and thrombin could mediate the neurite outgrowth activity of the factor (Monard, 1987, 1988). These considerations prompted the present studies, which included a n examination of the effects of free PN-1 and PN-1:thrombin complexes on neuroblastoma cells. Our data support the conclusion that the neurite outgrowth activity of PN-1 on neuroblastoma cells results from inhibiting thrombin and blocking thrombinmediated retraction of neurites.
Antibody production DIP-thrombin was prepared by reacting thrombin with diisopropylfluorophosphate (Sigma, St. Louis) as described (Low and Cunningham, 1982). The IgG fraction from serum of a rabbit immunized with the DIP-thrombin was purified using protein A-Sepharose affinity chromatography. The preparation and characterization of the mAbp-9 monoclonal antibody to human PN-1 is described in detail elsewhere (Wagner e t al., 1988).
Thrombin assay Thrombin was assayed by the procedure of Low and Cunningham (1982) with the following modifications. Only one incubation step was carried out, in which a sample of the conditioned medium was incubated with 5 ng l2'1-hirudin (8,000 cpmhg), 10 pg anti-DIPthrombin IgG, and 100 pg protein-A Sepharose in 1ml phosphate-buffered saline (pH 7.4) containing 1mgiml MATERIALS AND METHODS BSA. Following incubation for 2 hr at 37°C on a vertical shaker (60 rpm), the mixture was centrifuged a t Materials 8,000g for 6 min. The supernatants were gently aspiHighly purified human thrombin (110 Uinmole) rated and discarded without disturbing the protein-A (Fenton et al., 1977) was a kind gift from Dr. J.W. Sepharose pellets. One milliliter of 10 mM TRIS-HCl, Fenton I1 (Albany, NY). Human PN-1 was purified pH 8.6, 0.1% SDS, 0.05% Nonidet P-40 and 0.3N NaCl from serum-free culture medium conditioned by con- was added to each tube. The tubes were vigorously fluent human fibroblasts according to published proce- shaken for a few seconds and recentrifuged. Following dures that included a n immunopurification step (Van aspiration of the supernatants, the radioactivity in the Nostrand et al., 1988). Human anti-thrombin 111 pellets was determined in a gamma counter. Thrombin (ATIII), human urokinase, and leech hirudin were from standards were also assayed in parallel and used to Calbiochem (San Diego, CA). Thrombin was titrated as calculate the amounts of active thrombin in the samdescribed by Chase and Shaw (1969) and was ples (run in triplicate). The background radioactivity subsequently used to titrate PN-1 and ATIII using the was determined by carrying out the same protocol chromogenic thrombin substrate tosyl-Gly-Pro-Arg-4- without thrombin, and the observed radioactivity was nitroanilide-acetate (Chromozyme-TH; Boehringer- subtracted from all other readings. This simplified protocol enabled us to process a large number of samples in parallel; i t was as sensitive (detecting 10 pg thrombin) and as linear (up to 5 ng thrombinitube) a s the Fig. 1. Photomicrographs of neuroblastoma cells. Two days after original, more laborious, protocol. It should be emphaplating the cells in DME medium containing 10% FCS, the medium sized that anti-DIP-thrombin serum is inadequate for was replaced with fresh serum-free IIME medium containing the following additions: (A) none; (B) 4 nM PN-I; (C) 0.8 nM thrombin; (D) this assay, as it contains about 200 pg/ml ATIII, which interferes with the measurement of active thrombin; 0.8 nM thrombin and 4 nM PN-1. The cells were photographed using phase microscopy 4 hr later. the IgG fraction of this serum was therefore used.
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NEURITE OUTGROWTH ACTIVITY OF PROTEASE NEXIN-1 TABLE 1. Inhibition of the neurite-promoting activity of PN-1 by an anti-PN-1 monoclonal antibody* Addition a. DME medium containing 0.8?6 FCS None 9 nM PN-1 9 nM PN-1 20 kgiml mAbp-9 0.2 Uiml hirudin 0.2 Uiml hirudin + 20 pg/ml mAbp-9 b. Serum-free DME medium None 20 pgiml mAbp-9 2.7 nM thrombin 2.7 nM thrombin + 20 d m l mAbp-9
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Cell culture Mouse neuroblastoma cells, line Nb2a, were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbeco's modified Eagle's medium (DME medium; Gibco, Grand Island, NY) containing 10% fetal calf serum (FCS; Gibco) in an atmosphere of 95% air, 5% COz at 37°C as described earlier (Gurwitz and Cunningham, 1988). For experiments in serum-free medium, the serum-containing medium was aspirated and replaced with DME medium containing 1mgiml BSA. Inclusion of BSA in the serum-free medium was crucial to avoid loss of added PN-1 due to sticking to the culture dish surface. The cells were washed 3 times in serum-free medium to ensure complete removal of serum proteins. For neurite outgrowth assays, cells were plated a t a density of 20,000 cells per well in 12-well plates (Corning, 24 mm diameter). Neurite outgrowth was assayed as described previously (Gurwitz and Cunningham, 1988). Cells were scored positive for the presence of neurites when they had a t least one extension longer than one cell diameter. Values shown are means of duplicate assays. RESULTS Effects of PN-1 on neurite outgrowth under serum-free conditions in the presence and absence of thrombin The first step in determining the required interactions of PN-1 with other molecules or with neuroblastoma cells that bring about neurite outgrowth was to determine if free, uncomplexed PN-1 stimulated neu-
Fig. 3. A Purity of PN-1:thrombin complexes. '"'1-thrombin (14 nM) was incubated for 30 min at 37°C in a volume of 100 pl phosphatebuffered saline with the following additions: (lane 1) 10 nM PN-1; (lane 2) none; (lane 3) 30 nM PN-1:thrombin complexes. Samples were analyzed on a 10% acrylamide gel according to Laemmli (1970) and exposed to Kodak X-OMAT film. Lane 4 shows a silver-stained gel on which 1 pg of PN-1:thrombin complex was loaded. The molecular weight markers were phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase. E D Photomicrographs of neuroblastoma cells. Two days after plating the cells in DME medium containing 10%FCS, the medium was replaced with fresh DME medium containing 0.8% FCS and the following additions: (B) none; (C) 9 nM PN-1; (D) 12 nM PN-1:thrombin complexes. The cells were photographed 4 h r later.
Fig. 4. Effect of PN-1:thrombin complexes on neurite outgrowth. The medium on neuroblastoma cells was changed to DME medium containing 0.8% FCS and the indicated concentrations of PN-1 (0)or PN-1:thrombin complexes (*I. Cells were scored for neurite outgrowth 4 hr later.
rite outgrowth. These experiments were conducted under serum-free conditions to maximize the opportunity to examine the direct interaction of PN-1 with cells. AS reported earlier, neuroblastoma cells extend neurites when placed in serum-free medium (Seeds et al., 1970; Schubert et al., 1971, 1974). Under these conditions, addition of PN-1 had no detectable effect on the extent of neurite outgrowth (Fig. la,b). Similar results were obtained using a broad concentration range of PN-1: there were no detectable differences in the appearance of neuroblastoma cells in serum-free medium with or without PN-1. Previous studies showed that neurite outgrowth that occurs in serum-free medium can be blocked by added thrombin (Fig. 1C) (Gurwitz and Cunningham, 1988). Addition of excess PN-1 to the thrombin-containing cultures stimulated neurite outgrowth (Fig. 1D). Thus, in serum-free medium PN-1 stimulated neurite extension in the presence, but not in the absence, of added thrombin. Experiments were also conducted that quantitatively supported the above conclusions (Fig. 2). Neurite outgrowth was assayed as previously described (Gurwitz and Cunningham, 1988); cells were scored positive for the presence of neurites when they had at least one extension longer than one cell diameter. As shown, the rate and extent of neurite extension in serum-free medium were equal in the presence or absence of PN-1. Also, this rate of neurite outgrowth was equal to the rate of neurite outgrowth produced by adding PN-1 to cells whose neurite outgrowth had been blocked by added thrombin. Finally, the rate and extent of neurite retraction by added thrombin was equal in cells whose neurites had been extended in serum-free medium in the presence or absence of added PN-1 (Fig. 2).
Effects of an anti-PN-1monoclonal antibody on neurite outgrowth The above results suggest that PN-1 stimulates neurite outgrowth in neuroblastoma cells solely by inhibiting thrombin. To examine this further, we employed a n anti-PN-1 monoclonal antibody, which blocks its ca-
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Fig. 5. Effect of ATIII and heparin on neurite outgrowth and thrombin concentration in cultures of neuroblastoma cells. The medium on neuroblastoma cells was changed to serum-free DME medium supplemented with 0.8 nM thrombin. The indicated concentrations of heparin without ATIII (0)or with 1.2 nM ATIII ( 0 ) were added immediately following the medium change. A: Four hr later the media were removed and subsequently assayed for residual active thrombin as described in “Materials and Methods.” B: Following removal of the media, cells were fixed and scored for neurite outgrowth.
pacity to complex and inhibit serine proteinases (Wagner et al., 1988). This monoclonal antibody completely blocked the neurite-promoting capacity of PN-1 (Table 1).This was due to a specific interaction with the added PN-1 since the antibody did not interfere with neurite outgrowth induction by hirudin, a specific thrombin inhibitor previously shown to possess neurite-promoting activity (Monard et al., 1983; Gurwitz and Cunningham, 1988). In addition, it did not interfere with neurite outgrowth induced by placing the neuroblastoma cells in serum-free medium, or with the capacity of thrombin to inhibit neurite outgrowth under these conditions (Table 1).These observations demonstrate that PN-1 loses its neurite-promoting capacity when its thrombin inhibitory activity is blocked.
Effects of PN-1:thrombin complexes on neurite outgrowth In view of suggestions that the neurite outgrowth activity of the glial-derived nexin/PN-1 might be due to complexes it forms with thrombin (Monard, 1987, 19881, we directly evaluated the ability of PN1:thrombin complexes to stimulate neurite outgrowth. Pure PN-1:thrombin complexes were prepared as described in “Materials and Methods.” Their purity was verified by the identification of a single band on silverstained gels. The absence of detectable free PN-1 in the
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Fig. 6. Effect of PN-1 on neurite outgrowth and thrombin concentration in cultures of neuroblastoma cells. The medium on neuroblastoma cells was changed to serum-free DME medium containing 0.8 nM thrombin and the indicated concentrations of PN-1. Residual active thrombin and neurite outgrowth were determined as described in Figure 5.
preparation was demonstrated by the lack of formation of labeled PN-1 thrombin complexes after incubation with ‘261-thrombin (Fig. 3A). In addition, no residual thrombin activity could be detected in the preparation of PN-1:thrombin complexes using the extremely sensitive thrombin assay described in “Materials and Methods,” which detects 10 pg thrombin. About 1,000fold higher thrombin concentration is required to block neurite outgrowth (Gurwitz and Cunningham, 1988). The ability of these complexes to induce neurite extension was examined under conditions similar to those routinely used t o study the neurite-promoting potency of PN-1, i.e., in the presence of 0.8%’fetal calf serum. As shown in Figure 3 E D , such complexes did not promote neurite outgrowth, unlike free PN-1 tested under similar conditions. This conclusion was supported by the quantitative data of Figure 4. While free PN-1 produced half-maximal and maximal stimulation of neurite outgrowth a t 1.8 and 9 nM, respectively, PN-1: thrombin complexes were inactive at these concentrations. Similar results were obtained using PN-1:urokinase complexes (data not shown).
Effects of anti-thrombin I11 and heparin on neurite outgrowth We recently showed that heparin can induce neurite outgrowth in neuroblastoma cells maintained in serum-containing medium (Gurwitz and Cunningham, 1988).It was suggested that this was due to the ability of heparin to accelerate the inactivation of thrombin by
NEURITE OUTGROWTII ACTlVlTY OF PHOTEASE NEXIN-1
antithrombin I11 (ATIII). To examine this hypothesis, we studied the effects of ATIII and heparin on neurite outgrowth in serum-free medium that contained 0.8 nM thrombin to maintain the neuroblastoma cells in their undifferentiated state (Fig. 5). This thrombin concentration was chosen since it is equal to the amount detected in medium containing 10% serum, which is routinely used to culture these cells iGurwitz and Cunningham, 1988). Addition of heparin (up to 10 Fgiml) did not promote neurite outgrowth. In addition, it did not affect thrombin levels judged by measurements of thrombin activity (Fig. 5). Interestingly, in the absence of heparin addition of ATIII in excess over thrombin also did not stimulate neurite outgrowth and only marginally reduced the level of thrombin detected in the medium (Fig. 5). However, a combination of ATIII and heparin promoted neurite outgrowth, and this was accompanied by a large decrease in thrombin activity (Fig. 5 ) . Similar studies using PN-1 instead of ATIII revealed that it could stimulate neurite outgrowth in the absence of added heparin (Fig. 6). Moreover, PN-1 in the absence of added heparin caused large reductions in thrombin activity (Fig. 6). Note that for both PN-1 and ATIII a reduction of thrombin levels to less than 0.2 nM was required to observe a n increase in the percentage of cells extending neurites; a reduction of thrombin levels to less than 0.02 nM brought about maximal extension of neurites. These data indicate that the neurite-promoting activity of PN-1 and ATIII is dependent on their capacity to inhibit thrombin.
DISCUSSION The present studies were conducted on neuroblastoma cells since they are a n appropriate model system to study factors regulating differentiation of neurons. Although these immortalized cells do not form synapses, they extend neurites in response to certain factors. They also synthesize and release neurotransmitters and express surface receptors found on neurons (Schubert et al., 1974). A major advantage of studying cloned neuroblastoma cell lines is the absence of contaminating cell types. This is a n unavoidable problem with primary neuronal cultures where i t must be established that a given agent acts directly on neurons and not via glia. Moreover, neuroblastoma cells have been used in most of the prior studies t h a t established the neurite outgrowth activity of the glial-derived nexin/PN-1. A recent report indicated that the glial derived nexin also stimulates neurite outgrowth in serum-free cultures of chick sympathetic neurons. However, a 50-fold higher concentration of the factor was required t o stimulate neurite outgrowth compared to neuroblastoma cells (Zurn et al., 1988). Our studies were designed to address the basis of the neurite-promoting activity of the glial-derived nexini PN-1. The major conclusion of the present studies is that the neurite outgrowth activity of PN-1 on neuroblastoma cells depends on its ability to inhibit thrombin. PN-1 had no detectable neurite-promoting activity in the absence of thrombin, where neurite outgrowth occurs spontaneously (Fig. 1).Moreover, in serum-free medium thrombin added in excess over PN-1 retracted neurites, while PN-1 added in excess over thrombin led to neurite extension. The retraction of neurites by
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thrombin was identical in neuroblastoma cells whose neurites had been extended by placing the cells in a serum-free medium with or without the addition of PN1 (Fig. 2). An anti-PN-1 monoclonal antibody that blocks the ability of PN-1 to form complexes with and inhibit proteases blocked the ability of PN-1 to stimulate neurite outgrowth without inhibiting the neuritepromoting activity of another thrombin inhibitor, hirudin (Table 1).Studies on both PN-1 and ATIII showed that their neurite outgrowth activity was quantitatively related to their ability to inhibit thrombin (Figs. 5,6). Finally, the inability of PN-1:thrombin complexes to stimulate neurite outgrowth (Figs. 3,41 further supports the conclusion that the neurite outgrowth activity of PN-1 results from inhibiting thrombin. This conclusion is consistent with previous studies that showed that thrombin retracts neurites in neuroblastoma cells. The neurite retraction was specific for thrombin since it did not occur with higher concentrations of plasmin, urokinase, or trypsin (Gurwitz and Cunningham, 1988). It recently has been shown that PN-1 binds to the extracellular matrix of cultured fibroblasts (Farrell et al., 1988) and that this regulates both the activity and target protease specificity of PN-1. Fixed fibroblasts as well as extracellular matrix prepared from unfixed cells accelerate the inactivation of thrombin by PN-1; this is due mostly to heparan sulfate (Farrell and Cunningham, 1986, 1987). Moreover, binding of PN-1 to fixed fibroblasts o r their extracellular matrix blocks its ability to inhibit both urokinase and plasmin (Wagner et al., 1989). Similar results have been obtained with mouse neuroblastoma cells and human glioma cells (S. Wagner. A. Lau and D. Cunningham, unpublished results). Thus, PN-1 appears to be primarily a thrombin inhibitor when bound to cells. This cell-conferred specificity of PN-1 is in concert with the conclusion of the present studies that its neurite outgrowth activity depends on thrombin inhibition. We previously showed that heparin promotes neurite outgrowth in neuroblastoma cells maintained in a serum-containing medium (Gurwitz and Cunningham, 1988). This was interpreted to be due to inactivation of serum thrombin by serum ATIII, a reaction that is accelerated by heparin. The present studies support this hypothesis by showing that heparin by itself or ATIII by itself was incapable of stimulating neurite outgrowth in a serum-free medium that contained thrombin, but that neurite outgrowth occurred when the heparin and ATIII were added together. Moreover, there was a quantitative relationship in this experiment between inactivation of thrombin and stimulation of neurite outgrowth (Fig. 5). Under similar experimental conditions, PN-1 stimulated neurite outgrowth, and here also, there was a quantitative relationship between thrombin inactivation and neurite outgrowth (Fig. 6). The glial-derived nexin/PN-1 is present in brain (Reinhard et al., 1988), but its physiological function there remains to be identified. Its ability to stimulate neurite outgrowth in culture in the presence of thrombin does not necessarily imply that it is a neurotrophic factor. Likewise, the present observations on the neurite-promoting activity of ATIII do not imply a n in vivo neurotrophic function for this plasma protein. Apparently
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PN-1 is synthesized in the brain by astrocytes, since primary cultures of these cells synthesize and secrete PN-1 (Rosenblatt et al., 1987). We recently found that thrombin converts cultured stellate astrocytes to a nonstellate form, and that PN-1 can reverse this. As with the ability of thrombin to retract neurites on neuroblastoma cells, the conversion of astrocytes t o a nonstellate form was highly specific for thrombin and did not occur with other proteases tested (K. Cavanaugh, D. Gurwitz, D. Cunningham, R. Bradshaw, unpublished data). It is presently not clear whether thrombin or a thrombin-like protease is present in brain under normal conditions, or whether it occurs only after injury or breakdown of the blood brain barrier. Information about the distribution of PN-1 and its target proteinase(s) throughout the brain as well as the factors that control their synthesis, secretion, and turnover is also very sketchy. Experiments that address these points should provide important clues about the physiological roles of PN-1.
ACKNOWLEDGMENTS This work was supported by NIH grants GM 31609 and CA 12306. D.G. was supported by a Postdoctoral Fellowship from the University of California Cancer Research Coordinating Committee. We thank William Van Nostrand for active-site titration of thrombin and Steven Wagner and Alice Lau for the preparations of PN-1 and the monoclonal antibody mAbp-9. LITERATURE CITED Baker, J.B., Low, D.A., Simmer, R.L., and Cunningham, D.1). (1980) Protease-nexin: A cellular component that links thrombin and plasminogen activator and mediates their binding to cells. Cell, 21: 37-45. Chase, T., and Shaw, E. (1969) Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters: Titration of the enzymes. Biochemistry, 8r2212-2224. Farrell, D.H., and Cunningham. D.D. (1986) Human fibroblasts accelerate the inhibition of thrombin by protease nexin. Proc. Natl. Acad. Sci. U.S.A., 83:6858-6862. Farrell, D.H., and Cunningham, D.D. (1987) Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1. Biochem. J.,245.543-550. Farrell, D.H., Wagner, S.L., Yuan, R.H., and Cunningham, D.D. (1988) Localization of protease nexin-1 on the fibroblast extracellular matrix. J . Cell. Physiol., 134t179-188. Fenton, J.W., 11, Fasco, M.J., Stackrow, A.B., Aronson, D.L., Young, A.M., and Finlayson, J.S. (1977) Human thrombins. Production, evaluation, and properties of alpha-thrombin. J. Biol. Chem., 252: 3587-3598. Gloor, S., Odink, K., Guenther, J., Nick, H., and Monard, D. 11986) A glia-derived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell, 47:687-693. Guenther, J., Nick, H., and Monard, D. (1985) A glia-derived neuritepromoting factor with protease inhibitory activity. EMBO J., 4: 1963-1966. Gurwitz, D., and Cunningham, D.D. (1988) Thrombin modulates and
reverses neuroblastoma neurite outgrowth. Proc. Natl. Acad. Sci. U.S.A., 85:3440-3444. Hawkins, R.L., and Seeds, N.W. (1986) Effect of proteases and their inhibitors on neurite outgrowth from neonatal mouse sensory ganglia in culture. Brain Res., 398:63-70. Hawkins, R.L., and Seeds, N.W. (1989) Protease inhibitors influence the direction of neurite outgrowth. Dev. Brain Res., 45:203-209. Krystosek, A,, and Seeds, N.W. (1981) Plasminogen activator release at the neuronal growth cone. Science, 213:1532-1534. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227t680-685. Low, D.A., Baker, J.B., Koonce, W.C., and Cunningham, D.D. (1981) Released protease-nexin regulates cellular binding, internalization and degradation of serine proteases. Proc. Natl. Acad. Sci. U.S.A., 78t2340-2344. Low, D.A., and Cunningham, D.D. (1982) A novel method for measuring cell surface-hound thrombin: Detection o f iodination-induced changes in thrombin-binding affinity. J. Biol. Chem., 257:850-858. McGrogan, M., Gohari, J., Li, M., Hsu, C., Scott, R.W., Simonsen, C.C., and Baker, J.B. (1988) Molecular cloning and expression of two forms of human protease nexin 1. Bio/Technology, 6r172-177. Monard, D. (1987) Role of protease inhibition in cellular migration and neuritic growth. Biochem. Pharmacol., 36t1389-1392. Monard, D. (1988) Cell-derived proteases and protease inhibitors as regulators of neurite outgrowth. Trends Neurosci., 11.541-544. Monard. D.. Nidav. E.. Limat. A,. and Solomon. F. (1983) Inhibition of protease activity can lead 'to neurite extension in neuroblastoma cells. Prog. Brain Res., 58t359-364. Pittman, R.N., and Patterson, P.H. (1987) Characterization of an inhibitor of neuronal plasminogen activator released by heart cells. J . Neurosci., 7t2664-2673. Reinhard, E., Meier, R., Halftcr, W., Rovelli, G . , and Monard, D. (1988) Detection of glia-derived nexin in the olfactory system of the rat. Neuron, It387-394. Rosenblatt, D.E., Cotman, C.W., Nieto-Sapedro, M., Rowe, J.W., and Knauer, D.J. (1987) Identification of a protease inhibitor produced by astrocytes that is structurally and functionally homoiogous to human protease nexin-I. Brain Res., 4I5t40-48. Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J.H., Culp, W., and Brandt, B.L. (1974) Clonal cell lines from the rat central nervous system. Nature, 249: 224-227. Schubert, D., Humphreys, S., De Vitry, F., and Jacob, F. (1971) Induced differentiation of a neuroblastoma. Dev. B i d , 25.514-546. Schurch-Ratgeb, Y., and Monard, D. (1978) Brain development influences the appearance of glial factor-like activity in rat brain primary cultures. Nature, 273t308-310. Scott, R.W., Bergman, B.L., Bajpai, A,, Hersh, H.T., Rodriguez, H., Jones, B.N., Barreda, C., Watts, S., and Baker, J.B. (1985) Protease nexin: Properties and a modified purification procedure. J. Biol. Chem., 260:7029 -7034. Seeds, N.W., Gilman, A.G., Amano, T., and Nirenberg, M.W. (1970) Regulation of axon formation by clonal lines of a neural tumor. Proc. Natl. Acad. Sci. U.S.A., 66t160-167. Van Nostrand, W.E., Wagner, S.L., and Cunningham, D.D. (1988) Purification of a form of protease nexin 1 that hinds heparin with a low affinity. Biochemistry, 27r2176-2181. Wagner, S.L., Lau, A.L., and Cunningham, D.D. (1989) Binding of protease nexin-1 t o the fibroblast surface alters its target proteinase specificity. J. Biol. Chem., 2643311-615. Wagner, S.L., Van Nostrand, W.E., Lau, A.L., and Cunningham, D.D. (1988) Monoclonal antibodies to protease nexin-1 that differentially block its inhibition of target proteases. Biochemistry, 27.91732176. Zurn, A.D., Nick, H., and Monard, D. (1988) A glia-derived nexin promotes neurite outgrowth in cultured chick sympathetic neurons. Dev. Neurosci., 10:17-24.
Neurite Outgrowth Activity of Protease Nexin-I on Neuroblastoma Cells Requires Thrombin Inhibition DAVID GURWITZ AND DENNIS D. CUNNINGHAM' Department of Microbiology and Molecular Cenetirs, College of Medicine, University of California, Irvine, CA 92717 Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1 :thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-I, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-I did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in (he presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin Ill, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin I l l and PN-I corresponded to their inhibition of thrombin. Together, [hew observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.
There is much evidence that several protease inhibitors can stimulate neurite outgrowth from certain cultured neuroblastoma cells and primary neurons (Monard et al., 1983; Guenther et al., 1985; Hawkins and Seeds, 1986; Pittman and Patterson, 1987; Gurwitz and Cunningham, 1988; Zurn et al., 1988; Hawkins and Seeds, 1989). This has led to the suggestion that a balance between proteases and protease inhibitors may be critical for the regulation of neurite outgrowth (Monard, 1987, 1988; Pittman and Patterson, 1987). Two proteases that have been implicated in this control are plasminogen activator and thrombin. Plasminogen activator is released from the growth cones of cultured neurons and could facilitate neurite outgrowth (Krystosek and Seeds, 1981). It has been suggested that thrombin could play an important role in this regulation since several thrombin inhibitors stimulate outgrowth (Monard et al., 1983) and since thrombin itself retracts neurites (Gurwitz and Cunningham, 1988). A protease inhibitor that could play a physiological role in the regulation of neurite outgrowth is the glial-derived neurite-promoting factor or glial-derived nexin. Previous studies showed that mouse neuroblastoma cells and chick sympathetic neurons extend neurites in response to this factor (Schurch-Ratgeb and Monard, 1978; Monard et al., 1983; Zurn et al., 1988; Gurwitz and Cunningham, 1988). This factor was re1990 WILEY-LISS, INC.
cently shown to be identical with protease nexin-1 (PN1)(Gloor et al., 1986; McGrogan et al., 19881, which is secreted by a variety of cultured cells and rapidly inhibits thrombin, urokinase, and plasmin by forming complexes with their catalytic site serine residues (Baker et al., 1980; Scott et al., 1985). The complexes bind back to the cells and are rapidly internalized and degraded (Low et al., 1981). It has been suggested that the neurite outgrowth activity of the glial-derived nexiniPN-1 depends on inhibition of thrombin since hirudin, another thrombin inhibitor, also stimulates neurite outgrowth in neuroblastoma cells (Monard et al., 1983; Gurwitz and Cunningham, 1988). Thrombin blocks the neurite outgrowth activity of the glial-derived nexin/PN-1 and also brings about retraction of neurites (Gurwitz and Cunningham, 1988). Urokinase also blocks the neurite outgrowth activity of the glialderived nexin/PN-1, although this could be due t o its ability to form complexes with PN-1 and thus reduce the concentration of free PN-1. Urokinase, unlike thrombin, does not bring about retraction of neurites (Gurwitz and Cunningham, 1988).
Received June 19, 1989; accepted September 14, 1989. *To whom reprint requestsicorrespondence should be addressed.
NEURITE OUTGROWTH ACTIVITY OF PROTEASE NEXIN-1
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Mannheim, Indianapolis). Porcine heparin (168 Uimg), bovine serum albumin (BSA, RIA grade), and protein A-Sepharose were from Sigma Chemical Go. (St. Louis). Na1251was from New England Nuclear (Boston). It was used to radioiodinate hirudin and thrombin using Iodogen (Pierce, Rockford, IL) (Baker e t al., 1980). Electrophoresis reagents and molecular weight markers were from Bio-Rad (Richmond, CA).
Preparation of pure PN-1:thrombin complexes Thrombin (1.4 pm) was incubated with 7 pm PN-1 for 2 h r a t 23°C. The unreacted PN-1 was removed by incubating the mixture with protein A-Sepharose coupled to a monoclonal antibody, which binds free but not 0 2 4 6 complexed PN-1 (Wagner et al., 1988). After incubation for 4 h r a t room temperature with shaking, the time ( h l mixture was centrifuged at 8,000g for 15 min, and the Fig. 2. Effect of PN-1 on neurite outgrowth in the absence of added complexes were recovered in the supernatant. The puthrombin; effect of subsequent. thrombin addition. The medium on rity of the complexes was verified by SDS polyacrylaneuroblastoma cells was changed to serum-free DME medium with mide gel electrophoresis (Laemmli, 1970). PN-1:urokieither no addition (01, 4 nM PN-1 (*),or 0.8 nM thrombin (A,. At the nase complexes were prepared using a similar protocol. times indicated by the arrows, 8 nM thrombin was added to the first two sets of cultures, while 4 nM PN-1 was added to the third set. Cells were scored for neurite outgrowth at the indicated time points.
Although the above studies indicate a role for thrombin in the stimulation of neurite outgrowth by the glial-derived nexiniPN-1, there are important unanswered questions regarding the molecules that interact with cells to produce this response. For example, it was recently suggested that complexes between the glialderived nexiniPN-1 and thrombin could mediate the neurite outgrowth activity of the factor (Monard, 1987, 1988). These considerations prompted the present studies, which included a n examination of the effects of free PN-1 and PN-1:thrombin complexes on neuroblastoma cells. Our data support the conclusion that the neurite outgrowth activity of PN-1 on neuroblastoma cells results from inhibiting thrombin and blocking thrombinmediated retraction of neurites.
Antibody production DIP-thrombin was prepared by reacting thrombin with diisopropylfluorophosphate (Sigma, St. Louis) as described (Low and Cunningham, 1982). The IgG fraction from serum of a rabbit immunized with the DIP-thrombin was purified using protein A-Sepharose affinity chromatography. The preparation and characterization of the mAbp-9 monoclonal antibody to human PN-1 is described in detail elsewhere (Wagner e t al., 1988).
Thrombin assay Thrombin was assayed by the procedure of Low and Cunningham (1982) with the following modifications. Only one incubation step was carried out, in which a sample of the conditioned medium was incubated with 5 ng l2'1-hirudin (8,000 cpmhg), 10 pg anti-DIPthrombin IgG, and 100 pg protein-A Sepharose in 1ml phosphate-buffered saline (pH 7.4) containing 1mgiml MATERIALS AND METHODS BSA. Following incubation for 2 hr at 37°C on a vertical shaker (60 rpm), the mixture was centrifuged a t Materials 8,000g for 6 min. The supernatants were gently aspiHighly purified human thrombin (110 Uinmole) rated and discarded without disturbing the protein-A (Fenton et al., 1977) was a kind gift from Dr. J.W. Sepharose pellets. One milliliter of 10 mM TRIS-HCl, Fenton I1 (Albany, NY). Human PN-1 was purified pH 8.6, 0.1% SDS, 0.05% Nonidet P-40 and 0.3N NaCl from serum-free culture medium conditioned by con- was added to each tube. The tubes were vigorously fluent human fibroblasts according to published proce- shaken for a few seconds and recentrifuged. Following dures that included a n immunopurification step (Van aspiration of the supernatants, the radioactivity in the Nostrand et al., 1988). Human anti-thrombin 111 pellets was determined in a gamma counter. Thrombin (ATIII), human urokinase, and leech hirudin were from standards were also assayed in parallel and used to Calbiochem (San Diego, CA). Thrombin was titrated as calculate the amounts of active thrombin in the samdescribed by Chase and Shaw (1969) and was ples (run in triplicate). The background radioactivity subsequently used to titrate PN-1 and ATIII using the was determined by carrying out the same protocol chromogenic thrombin substrate tosyl-Gly-Pro-Arg-4- without thrombin, and the observed radioactivity was nitroanilide-acetate (Chromozyme-TH; Boehringer- subtracted from all other readings. This simplified protocol enabled us to process a large number of samples in parallel; i t was as sensitive (detecting 10 pg thrombin) and as linear (up to 5 ng thrombinitube) a s the Fig. 1. Photomicrographs of neuroblastoma cells. Two days after original, more laborious, protocol. It should be emphaplating the cells in DME medium containing 10% FCS, the medium sized that anti-DIP-thrombin serum is inadequate for was replaced with fresh serum-free IIME medium containing the following additions: (A) none; (B) 4 nM PN-I; (C) 0.8 nM thrombin; (D) this assay, as it contains about 200 pg/ml ATIII, which interferes with the measurement of active thrombin; 0.8 nM thrombin and 4 nM PN-1. The cells were photographed using phase microscopy 4 hr later. the IgG fraction of this serum was therefore used.
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NEURITE OUTGROWTH ACTIVITY OF PROTEASE NEXIN-1 TABLE 1. Inhibition of the neurite-promoting activity of PN-1 by an anti-PN-1 monoclonal antibody* Addition a. DME medium containing 0.8?6 FCS None 9 nM PN-1 9 nM PN-1 20 kgiml mAbp-9 0.2 Uiml hirudin 0.2 Uiml hirudin + 20 pg/ml mAbp-9 b. Serum-free DME medium None 20 pgiml mAbp-9 2.7 nM thrombin 2.7 nM thrombin + 20 d m l mAbp-9
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0
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Cell culture Mouse neuroblastoma cells, line Nb2a, were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbeco's modified Eagle's medium (DME medium; Gibco, Grand Island, NY) containing 10% fetal calf serum (FCS; Gibco) in an atmosphere of 95% air, 5% COz at 37°C as described earlier (Gurwitz and Cunningham, 1988). For experiments in serum-free medium, the serum-containing medium was aspirated and replaced with DME medium containing 1mgiml BSA. Inclusion of BSA in the serum-free medium was crucial to avoid loss of added PN-1 due to sticking to the culture dish surface. The cells were washed 3 times in serum-free medium to ensure complete removal of serum proteins. For neurite outgrowth assays, cells were plated a t a density of 20,000 cells per well in 12-well plates (Corning, 24 mm diameter). Neurite outgrowth was assayed as described previously (Gurwitz and Cunningham, 1988). Cells were scored positive for the presence of neurites when they had a t least one extension longer than one cell diameter. Values shown are means of duplicate assays. RESULTS Effects of PN-1 on neurite outgrowth under serum-free conditions in the presence and absence of thrombin The first step in determining the required interactions of PN-1 with other molecules or with neuroblastoma cells that bring about neurite outgrowth was to determine if free, uncomplexed PN-1 stimulated neu-
Fig. 3. A Purity of PN-1:thrombin complexes. '"'1-thrombin (14 nM) was incubated for 30 min at 37°C in a volume of 100 pl phosphatebuffered saline with the following additions: (lane 1) 10 nM PN-1; (lane 2) none; (lane 3) 30 nM PN-1:thrombin complexes. Samples were analyzed on a 10% acrylamide gel according to Laemmli (1970) and exposed to Kodak X-OMAT film. Lane 4 shows a silver-stained gel on which 1 pg of PN-1:thrombin complex was loaded. The molecular weight markers were phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase. E D Photomicrographs of neuroblastoma cells. Two days after plating the cells in DME medium containing 10%FCS, the medium was replaced with fresh DME medium containing 0.8% FCS and the following additions: (B) none; (C) 9 nM PN-1; (D) 12 nM PN-1:thrombin complexes. The cells were photographed 4 h r later.
Fig. 4. Effect of PN-1:thrombin complexes on neurite outgrowth. The medium on neuroblastoma cells was changed to DME medium containing 0.8% FCS and the indicated concentrations of PN-1 (0)or PN-1:thrombin complexes (*I. Cells were scored for neurite outgrowth 4 hr later.
rite outgrowth. These experiments were conducted under serum-free conditions to maximize the opportunity to examine the direct interaction of PN-1 with cells. AS reported earlier, neuroblastoma cells extend neurites when placed in serum-free medium (Seeds et al., 1970; Schubert et al., 1971, 1974). Under these conditions, addition of PN-1 had no detectable effect on the extent of neurite outgrowth (Fig. la,b). Similar results were obtained using a broad concentration range of PN-1: there were no detectable differences in the appearance of neuroblastoma cells in serum-free medium with or without PN-1. Previous studies showed that neurite outgrowth that occurs in serum-free medium can be blocked by added thrombin (Fig. 1C) (Gurwitz and Cunningham, 1988). Addition of excess PN-1 to the thrombin-containing cultures stimulated neurite outgrowth (Fig. 1D). Thus, in serum-free medium PN-1 stimulated neurite extension in the presence, but not in the absence, of added thrombin. Experiments were also conducted that quantitatively supported the above conclusions (Fig. 2). Neurite outgrowth was assayed as previously described (Gurwitz and Cunningham, 1988); cells were scored positive for the presence of neurites when they had at least one extension longer than one cell diameter. As shown, the rate and extent of neurite extension in serum-free medium were equal in the presence or absence of PN-1. Also, this rate of neurite outgrowth was equal to the rate of neurite outgrowth produced by adding PN-1 to cells whose neurite outgrowth had been blocked by added thrombin. Finally, the rate and extent of neurite retraction by added thrombin was equal in cells whose neurites had been extended in serum-free medium in the presence or absence of added PN-1 (Fig. 2).
Effects of an anti-PN-1monoclonal antibody on neurite outgrowth The above results suggest that PN-1 stimulates neurite outgrowth in neuroblastoma cells solely by inhibiting thrombin. To examine this further, we employed a n anti-PN-1 monoclonal antibody, which blocks its ca-
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Fig. 5. Effect of ATIII and heparin on neurite outgrowth and thrombin concentration in cultures of neuroblastoma cells. The medium on neuroblastoma cells was changed to serum-free DME medium supplemented with 0.8 nM thrombin. The indicated concentrations of heparin without ATIII (0)or with 1.2 nM ATIII ( 0 ) were added immediately following the medium change. A: Four hr later the media were removed and subsequently assayed for residual active thrombin as described in “Materials and Methods.” B: Following removal of the media, cells were fixed and scored for neurite outgrowth.
pacity to complex and inhibit serine proteinases (Wagner et al., 1988). This monoclonal antibody completely blocked the neurite-promoting capacity of PN-1 (Table 1).This was due to a specific interaction with the added PN-1 since the antibody did not interfere with neurite outgrowth induction by hirudin, a specific thrombin inhibitor previously shown to possess neurite-promoting activity (Monard et al., 1983; Gurwitz and Cunningham, 1988). In addition, it did not interfere with neurite outgrowth induced by placing the neuroblastoma cells in serum-free medium, or with the capacity of thrombin to inhibit neurite outgrowth under these conditions (Table 1).These observations demonstrate that PN-1 loses its neurite-promoting capacity when its thrombin inhibitory activity is blocked.
Effects of PN-1:thrombin complexes on neurite outgrowth In view of suggestions that the neurite outgrowth activity of the glial-derived nexin/PN-1 might be due to complexes it forms with thrombin (Monard, 1987, 19881, we directly evaluated the ability of PN1:thrombin complexes to stimulate neurite outgrowth. Pure PN-1:thrombin complexes were prepared as described in “Materials and Methods.” Their purity was verified by the identification of a single band on silverstained gels. The absence of detectable free PN-1 in the
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Fig. 6. Effect of PN-1 on neurite outgrowth and thrombin concentration in cultures of neuroblastoma cells. The medium on neuroblastoma cells was changed to serum-free DME medium containing 0.8 nM thrombin and the indicated concentrations of PN-1. Residual active thrombin and neurite outgrowth were determined as described in Figure 5.
preparation was demonstrated by the lack of formation of labeled PN-1 thrombin complexes after incubation with ‘261-thrombin (Fig. 3A). In addition, no residual thrombin activity could be detected in the preparation of PN-1:thrombin complexes using the extremely sensitive thrombin assay described in “Materials and Methods,” which detects 10 pg thrombin. About 1,000fold higher thrombin concentration is required to block neurite outgrowth (Gurwitz and Cunningham, 1988). The ability of these complexes to induce neurite extension was examined under conditions similar to those routinely used t o study the neurite-promoting potency of PN-1, i.e., in the presence of 0.8%’fetal calf serum. As shown in Figure 3 E D , such complexes did not promote neurite outgrowth, unlike free PN-1 tested under similar conditions. This conclusion was supported by the quantitative data of Figure 4. While free PN-1 produced half-maximal and maximal stimulation of neurite outgrowth a t 1.8 and 9 nM, respectively, PN-1: thrombin complexes were inactive at these concentrations. Similar results were obtained using PN-1:urokinase complexes (data not shown).
Effects of anti-thrombin I11 and heparin on neurite outgrowth We recently showed that heparin can induce neurite outgrowth in neuroblastoma cells maintained in serum-containing medium (Gurwitz and Cunningham, 1988).It was suggested that this was due to the ability of heparin to accelerate the inactivation of thrombin by
NEURITE OUTGROWTII ACTlVlTY OF PHOTEASE NEXIN-1
antithrombin I11 (ATIII). To examine this hypothesis, we studied the effects of ATIII and heparin on neurite outgrowth in serum-free medium that contained 0.8 nM thrombin to maintain the neuroblastoma cells in their undifferentiated state (Fig. 5). This thrombin concentration was chosen since it is equal to the amount detected in medium containing 10% serum, which is routinely used to culture these cells iGurwitz and Cunningham, 1988). Addition of heparin (up to 10 Fgiml) did not promote neurite outgrowth. In addition, it did not affect thrombin levels judged by measurements of thrombin activity (Fig. 5). Interestingly, in the absence of heparin addition of ATIII in excess over thrombin also did not stimulate neurite outgrowth and only marginally reduced the level of thrombin detected in the medium (Fig. 5). However, a combination of ATIII and heparin promoted neurite outgrowth, and this was accompanied by a large decrease in thrombin activity (Fig. 5 ) . Similar studies using PN-1 instead of ATIII revealed that it could stimulate neurite outgrowth in the absence of added heparin (Fig. 6). Moreover, PN-1 in the absence of added heparin caused large reductions in thrombin activity (Fig. 6). Note that for both PN-1 and ATIII a reduction of thrombin levels to less than 0.2 nM was required to observe a n increase in the percentage of cells extending neurites; a reduction of thrombin levels to less than 0.02 nM brought about maximal extension of neurites. These data indicate that the neurite-promoting activity of PN-1 and ATIII is dependent on their capacity to inhibit thrombin.
DISCUSSION The present studies were conducted on neuroblastoma cells since they are a n appropriate model system to study factors regulating differentiation of neurons. Although these immortalized cells do not form synapses, they extend neurites in response to certain factors. They also synthesize and release neurotransmitters and express surface receptors found on neurons (Schubert et al., 1974). A major advantage of studying cloned neuroblastoma cell lines is the absence of contaminating cell types. This is a n unavoidable problem with primary neuronal cultures where i t must be established that a given agent acts directly on neurons and not via glia. Moreover, neuroblastoma cells have been used in most of the prior studies t h a t established the neurite outgrowth activity of the glial-derived nexin/PN-1. A recent report indicated that the glial derived nexin also stimulates neurite outgrowth in serum-free cultures of chick sympathetic neurons. However, a 50-fold higher concentration of the factor was required t o stimulate neurite outgrowth compared to neuroblastoma cells (Zurn et al., 1988). Our studies were designed to address the basis of the neurite-promoting activity of the glial-derived nexini PN-1. The major conclusion of the present studies is that the neurite outgrowth activity of PN-1 on neuroblastoma cells depends on its ability to inhibit thrombin. PN-1 had no detectable neurite-promoting activity in the absence of thrombin, where neurite outgrowth occurs spontaneously (Fig. 1).Moreover, in serum-free medium thrombin added in excess over PN-1 retracted neurites, while PN-1 added in excess over thrombin led to neurite extension. The retraction of neurites by
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thrombin was identical in neuroblastoma cells whose neurites had been extended by placing the cells in a serum-free medium with or without the addition of PN1 (Fig. 2). An anti-PN-1 monoclonal antibody that blocks the ability of PN-1 to form complexes with and inhibit proteases blocked the ability of PN-1 to stimulate neurite outgrowth without inhibiting the neuritepromoting activity of another thrombin inhibitor, hirudin (Table 1).Studies on both PN-1 and ATIII showed that their neurite outgrowth activity was quantitatively related to their ability to inhibit thrombin (Figs. 5,6). Finally, the inability of PN-1:thrombin complexes to stimulate neurite outgrowth (Figs. 3,41 further supports the conclusion that the neurite outgrowth activity of PN-1 results from inhibiting thrombin. This conclusion is consistent with previous studies that showed that thrombin retracts neurites in neuroblastoma cells. The neurite retraction was specific for thrombin since it did not occur with higher concentrations of plasmin, urokinase, or trypsin (Gurwitz and Cunningham, 1988). It recently has been shown that PN-1 binds to the extracellular matrix of cultured fibroblasts (Farrell et al., 1988) and that this regulates both the activity and target protease specificity of PN-1. Fixed fibroblasts as well as extracellular matrix prepared from unfixed cells accelerate the inactivation of thrombin by PN-1; this is due mostly to heparan sulfate (Farrell and Cunningham, 1986, 1987). Moreover, binding of PN-1 to fixed fibroblasts o r their extracellular matrix blocks its ability to inhibit both urokinase and plasmin (Wagner et al., 1989). Similar results have been obtained with mouse neuroblastoma cells and human glioma cells (S. Wagner. A. Lau and D. Cunningham, unpublished results). Thus, PN-1 appears to be primarily a thrombin inhibitor when bound to cells. This cell-conferred specificity of PN-1 is in concert with the conclusion of the present studies that its neurite outgrowth activity depends on thrombin inhibition. We previously showed that heparin promotes neurite outgrowth in neuroblastoma cells maintained in a serum-containing medium (Gurwitz and Cunningham, 1988). This was interpreted to be due to inactivation of serum thrombin by serum ATIII, a reaction that is accelerated by heparin. The present studies support this hypothesis by showing that heparin by itself or ATIII by itself was incapable of stimulating neurite outgrowth in a serum-free medium that contained thrombin, but that neurite outgrowth occurred when the heparin and ATIII were added together. Moreover, there was a quantitative relationship in this experiment between inactivation of thrombin and stimulation of neurite outgrowth (Fig. 5). Under similar experimental conditions, PN-1 stimulated neurite outgrowth, and here also, there was a quantitative relationship between thrombin inactivation and neurite outgrowth (Fig. 6). The glial-derived nexin/PN-1 is present in brain (Reinhard et al., 1988), but its physiological function there remains to be identified. Its ability to stimulate neurite outgrowth in culture in the presence of thrombin does not necessarily imply that it is a neurotrophic factor. Likewise, the present observations on the neurite-promoting activity of ATIII do not imply a n in vivo neurotrophic function for this plasma protein. Apparently
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PN-1 is synthesized in the brain by astrocytes, since primary cultures of these cells synthesize and secrete PN-1 (Rosenblatt et al., 1987). We recently found that thrombin converts cultured stellate astrocytes to a nonstellate form, and that PN-1 can reverse this. As with the ability of thrombin to retract neurites on neuroblastoma cells, the conversion of astrocytes t o a nonstellate form was highly specific for thrombin and did not occur with other proteases tested (K. Cavanaugh, D. Gurwitz, D. Cunningham, R. Bradshaw, unpublished data). It is presently not clear whether thrombin or a thrombin-like protease is present in brain under normal conditions, or whether it occurs only after injury or breakdown of the blood brain barrier. Information about the distribution of PN-1 and its target proteinase(s) throughout the brain as well as the factors that control their synthesis, secretion, and turnover is also very sketchy. Experiments that address these points should provide important clues about the physiological roles of PN-1.
ACKNOWLEDGMENTS This work was supported by NIH grants GM 31609 and CA 12306. D.G. was supported by a Postdoctoral Fellowship from the University of California Cancer Research Coordinating Committee. We thank William Van Nostrand for active-site titration of thrombin and Steven Wagner and Alice Lau for the preparations of PN-1 and the monoclonal antibody mAbp-9. LITERATURE CITED Baker, J.B., Low, D.A., Simmer, R.L., and Cunningham, D.1). (1980) Protease-nexin: A cellular component that links thrombin and plasminogen activator and mediates their binding to cells. Cell, 21: 37-45. Chase, T., and Shaw, E. (1969) Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters: Titration of the enzymes. Biochemistry, 8r2212-2224. Farrell, D.H., and Cunningham. D.D. (1986) Human fibroblasts accelerate the inhibition of thrombin by protease nexin. Proc. Natl. Acad. Sci. U.S.A., 83:6858-6862. Farrell, D.H., and Cunningham, D.D. (1987) Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1. Biochem. J.,245.543-550. Farrell, D.H., Wagner, S.L., Yuan, R.H., and Cunningham, D.D. (1988) Localization of protease nexin-1 on the fibroblast extracellular matrix. J . Cell. Physiol., 134t179-188. Fenton, J.W., 11, Fasco, M.J., Stackrow, A.B., Aronson, D.L., Young, A.M., and Finlayson, J.S. (1977) Human thrombins. Production, evaluation, and properties of alpha-thrombin. J. Biol. Chem., 252: 3587-3598. Gloor, S., Odink, K., Guenther, J., Nick, H., and Monard, D. 11986) A glia-derived neurite promoting factor with protease inhibitory activity belongs to the protease nexins. Cell, 47:687-693. Guenther, J., Nick, H., and Monard, D. (1985) A glia-derived neuritepromoting factor with protease inhibitory activity. EMBO J., 4: 1963-1966. Gurwitz, D., and Cunningham, D.D. (1988) Thrombin modulates and
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