Release of plasminogen activator and a calcium-dependent metalloprotease from cultured sympathetic and sensory neurons

DEVELOPMENTAL

BIOLOGY

110,

91-101

(19%)

Release of Plasminogen Activator and a Calcium-Dependent Metalloprotease from Cultured Sympathetic and Sensory Neurons N. PITTMAN’

RANDALL Division

of Biology

Received

216-76, October

California

Institute

1, 1984; accepted

of Technology,

in revised

form

Pasadena, January

91125

Califomzia

4, 1985

Cultures of neurons from neonatal rat superior cervical, dorsal root, and trigeminal ganglia were grown in the absence of nonneuronal cells in serum-free defined medium. Proteins metabolically labeled with radioactive amino acids and spontaneously released into the culture medium were studied using two-dimensional gel electrophoresis and photofluorography. All three populations of neurons released 12-15 major proteins into the culture medium. Four proteins were released selectively by sympathetic neurons and two proteins were consistently released by both populations of sensory neurons but not by sympathetic neurons. Enzymatic activities are associated with at least two of the released proteins. One is a calcium-dependent metalloprotease, and the other a plasminogen activator. The calcium-dependent metalloprotease has a MW of 62 kDa, requires millimolar calcium for maximum activity, and has a restricted substrate specificity. It degraded native and denatured collagen more readily than casein, albumin, or fibronectin and denatured collagen (gelatin) was a better substrate than native collagen. The plasminogen activator released by neurons has a MW of 51 kDa and is converted to an active 32 kDa form. Its physiochemical properties are similar to urokinase and it was precipitated by a rabbit antiserum produced against human urokinase. A large fraction of both proteases was released by distal processes and/or growth cones suggesting that these proteases could be involved in growth cone functions. 0 1986 Academic press, Inc.

et al., 1982; Soreq and Miskin,

1983) and histological assays have shown that neuroblastoma cells (Krystosek and Seeds, 1981a; Soreq et al., 1983) as well as sensory neurons (Krystosek and Seeds, 1984) release plasminogen activator. Proteases have also been implicated in Schwann cell proliferation (Kalderon, 1982), neurotransmission (Baxter et al, 1983), and memory storage (Lynch and Baudry, 1984). Neuronal cultures free of nonneuronal cells provide a good system to characterize proteins released by neurons. It was of interest to define the proteins released by sympathetic and sensory neurons and to determine if proteolytic activities were associated with these proteins. A preliminary account of this work has appeared (Pittman, 1983).

INTRODUCTION

Neuronal migration, neurite outgrowth, target recognition, and synaptogenesis are a few of the developmental events in which neurons may interact with as well as modify their local environment. The cellular and molecular bases of these processes are unknown, although direct cell-cell contact as well as the release of macromolecules by both neurons and nonneuronal cells are likely to be important (Varon and Adler, 1980; Patterson, 1982; Edelman, 1983; Barde et ah, 1983). Sympathetic neurons spontaneously release a number of glycoproteins which could serve important functions in neuronal development by interacting with the neuron’s local environment (Sweadner, 1981). One means of determining the function of proteins released by neurons is to determine whether specific enzymatic activities are associated with them. The consequences of altering these activities during development could then be assessed. The possibility that specific enzymatic or proteolytic activities may play a role in the developing nervous system has been investigated recently by a number of laboratories. The serine protease, plasminogen activator, has been implicated in granule cell migration in the cerebellum (Krystosek and Seeds, 1981b; Moonen ’ Present Pennsylvania

address: Department School of Medicine,

of Pharmacology Philadelphia,

G3, University Pa. 19104.

MATERIALS

AND

METHODS

Materials The following drugs and reagents were used: rabbit antiserum against human urokinase (Green Cross Corp., Osaka), rabbit antiserum against human plasminogen (Accurate Chemical, Westbury, N. Y.), fibronectin (Collaborative Research, Lexington, Mass.), fibrinogen (Calbiochem, San Diego, Calif.), protein A-Sepharose and lysine-Sepharose (Pharmacia, Piscataway, N. J.). The following were obtained from Sigma Chemical, St. Louis: gelatin, thrombin, N-ethylmaleamide, iodoacetate, 2-mercaptoethanol, pepstatin A, leupeptin, p-

of

91

0012-1606/85 Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

92

DEVELOPMENTAL BIOLOGY

nitrophenylguanidinobenzoate, 6-aminocaproic acid, benzamidine, soybean trypsin inhibitor, egg white trypsin inhibitor, phosphoramidon, CBZ-Gly-Pheamide, PMSF, DFP, trypsin, chymotrypsin, and urokinase. Plasminogen was purified from human serum using a lysine-Sepharose affinity column according to the method of Deutsch and Mertz (1970). Cell Culture

Neurons from the superior cervical, dorsal root, and trigeminal ganglia of l-day-old rats were dissociated into small clumps and single cells by treating with dispase (5 mg/ml) and collagenase (1 mg/ml) in phosphate-buffered saline containing 50 mM glucose for 30 min, followed by trituration using a 20-gauge needle. Occasionally, neurons from superior cervical ganglia were mechanically dissociated (Hawrot and Patterson, 1979). Cells were preplated in growth medium on tissue culture plastic for 1.5 hr to remove nonneuronal cells. Neurons were grown in L&CO2 medium containing rat serum and nerve growth factor (Hawrot and Patterson, 1979) for 9 days during which time nonneuronal cells were killed by treatment for 24 hr with 10 PM cytosine arabinoside on Days 1, 4, and 7 of culture. Cultures were then grown in a modification of the Nl serum-free defined medium (Bottenstein et al., 1979) containing L15-CO2 medium, stable and fresh vitamins (Hawrot and Patterson, 1979), insulin (5 pg/ml), transferrin (10 pg/ml), progesterone (20 nM), putrescine (100 p&f), selenium (30 p&f), and NGF (0.5 pg/ml) (see also: Wolinsky et ak, 1985). Occasionally, sympathetic neurons were grown in defined medium containing 20 mM KCl, and sensory neurons were routinely grown in this medium. Cultures 3-6 weeks old were used in all studies. Cultures of ganglionic nonneuronal cells were prepared either by maintaining the cells that attached following preplating of dissociated ganglia or by allowing nonneuronal cells to overgrow neuronal cultures for 2-3 weeks. Nonneuronal cells were cultured in the same medium as neurons. Metabolic Labeling of Released Proteins

Neurons (1000-3000) were grown in an 0.8-cm (diameter) well cut in the bottom of a 35-mm petri dish. Proteins were metabolically labeled with either 50 &i of [4,5-3H]leucine (40-60 Ci/mmole; New England Nuclear) or 50 &i of [35S]methionine (1000 Ci/mmole; New England Nuclear) in serum-free L15-CO2 medium containing 10% of the normal amount of leucine or methionine. Labeling was performed for 12-16 hr after which the medium (40 ~1) was removed from the

VOLUME 110,1985

cultures, mixed with an equal volume of either lysis buffer (O’Farrell et ak, 1977) or sample buffer (O’Farrell, 1975), and frozen at -70°C. Two-Dimensional Gel Electrophoresis and Photojuorography

Released proteins were analyzed in the first dimension either by isoelectric focusing (IEF) or by nonequilibrium pH gradient electrophoresis (NEPHGE) followed by electrophoresis in the second dimension on slab gels with a linear gradient of 5-15% acrylamide. IEF was performed with tube gels containing 2% pH 3.5-10 and 0.5% pH 7-9 ampholines according to the method of O’Farrell (1975). NEPHGE was performed with tube gels containing 2% pH 3.5-10 ampholines according to the method of O’Farrell et al. (1977). Sodium dodecyl sulfate (SDS) slab gel electrophoresis was carried out using the Laemmeli buffer system (1970) and gel photofluorography was performed by the method of Bonner and Laskey (1974). DuPont Cronex Lightning-Plus intensifying screens were used to enhance sensitivity. Determination of Protease Activity Gel Electrophoresis

following

Neurons (2000-4000) were grown in 24-well (2 cm’/ well) cluster dishes in 0.4 ml serum-free medium. Medium conditioned by neurons was collected every 24 or 48 hr and samples of 0.02-0.04 ml were used for measuring proteases. Therefore, the quantity of proteases released by approximately 200 neurons during a 48-hr period is easily detected with the standard procedure, and by using radiolabeled substrates (see below), detection of proteases released by a single neuron should be feasible. Proteases released by neurons were characterized following SDS slab gel electrophoresis using a modification of the method of Heussen and Dowdle (1980). Separating gels with 7.3% acrylamide, 0.31% bisacrylamide, and 0.11% gelatin were used. Plasminogen (25 pg/ml) was included in some gels in order to detect plasminogen activator activity. SDS gel electrophoresis was performed using the buffer system of Laemmli (1970) under nonreducing conditions at a constant current of 9 mA at 4°C. SDS (2% final concentration) was added to samples and following electrophoresis, was removed from the gel by washing three times with 15 vol of 2% Triton X100 (20-30 min each) while shaking at room temperature. The gel was rinsed three to four times (5 min each) in 20 mM Tris-HCl (pH 7.3 for the calciumdependent metalloprotease; pH 8.3 for plasmiogen activator or pH 7.7 for detection of both proteases) followed by incubation in the same buffer (* activators

RANDALL N. PITTMAN

or inhibitors) at 30°C for 12-24 hr. Gels were fixed and stained for 1 hr at room temperature using 0.1% amido black in acetic acid:methanol:water (1:3:6) and were destained with acetic acid:methanol:water (1:3:6). A modification of the gel electrophoresis assay was used to measure proteolysis of radiolabeled extracellular matrix components. Separating gels contained 9.6% acrylamide, 0.42% bisacrylamide, and 60 rig/ml of either [1251]fibronectin (1.6 &i), [3H]collagen (NEN; 0.3 &i), or [3H]gelatin (0.3 &i). [3H]Gelatin was prepared by heat denaturing (56°C for 30 min) commercially available [3H]collagen, and fibronectin (200 pg) was iodinated using 1 mCi Nalz51 and Iodogen (Pierce Chemical Co.). Gels containing [3H]collagen were poured at 4°C to avoid denaturation of collagen during polymerization. Following electrophoresis at 9 mA (4”C), SDS was removed using Triton X-100 and gels were incubated in 20 mM Tris-HCl (pH 7.3) containing 5 mM Ca2+ at 30°C for 12-15 hr. Gels were then fixed and either dried immediately ([lWI]fibronectin) or processed for photofluorography ([3H]collagen and rH]gelatin; Bonner and Laskey, 1974) followed by autoradiography.

New-ma1

93

Proteases

of sympathetic neurons; Mains and Patterson, 1973) cultures of sympathetic and sensory neurons exhibited obvious differences in appearance (Fig. 1). After 4 weeks of growth, cultures of sympathetic neurons had a more extensive outgrowth of processes and a more uniform distribution of cell diameters. Cultures of neurons from the superior cervical, dorsal root, and trigeminal ganglia all released 12-15 major proteins (Figs. 2a-c) as well as a few minor proteins (not visible in Fig. 2). Proteins spontaneously released by cultures of ganglionic nonneuronal cells were dis-

Quantitative Measurement of Plasminogen Activator Activity

Purified fibrinogen (5 mg) was iodinated with 1.25 mCi Nalz51 using 5 mg/ml chloramine-T and dialyzed exhaustively against phosphate-buffered saline. Precipitated protein was removed by centrifugation at 13,000g for 2 min. The resulting supernatant contained 2.5 X lo5 cpm/pg fibrinogen and 99.7% of the counts were precipitable with trichloroacetic acid. Plasminogen activator activity was measured using a modification of the method of Unkeless et al. (1973). [1251]Fibrinogen was diluted with unlabeled fibrinogen to a final concentration of 80 pug/ml (4-5 X lo5 cpm/ml). Twenty micrograms of fibrinogen (lo5 cpm) was added to each well of a 24-well cluster dish (2 cm2/well) and dried under vacuum at 40°C for 40 hr. Fibrinogen was converted to fibrin by adding 0.25 units of thrombin to each well for 1 hr at 37°C followed by washing five times with phosphate-buffered saline. Assays were run at 37°C in 0.5 ml of 0.1 M Tris-HCl, pH 8.1 containing 0.1% gelatin, 2 pg of human plasminogen, and 5-50 ~1 of released proteins. Aliquots were removed and radioactivity was determined after 1, 2, and 3 hr. RESULTS

Proteins Spontaneously Released by Cultures of Neurons and Nonneuronal Cells

When grown under identical conditions originally developed to maximize viability

(conditions and growth

FIG. 1. Phase superior 4 weeks

micrographs cervical (a), trigeminal in culture. X200.

of living cultures of neurons (b), and dorsal root (c) ganglia

from after

94

DEVELOPMENTAL

BIOLOGY

VOLUME

110, 1985

so-

3ctBASIC

se

e ACIDIC

BASIC

ACIDIC

FIG. 2. Two-dimensional autoradiograms of proteins metabolically labeled with [3H]leucine and spontaneously released into the culture medium by neurons from superior cervical (a), trigeminal (b), and dorsal root (c) ganglia and by nonneuronal cells from the trigeminal ganglion (d). Proteins selectively released by sympathetic neurons are circled and arrows indicate proteins selectively released by sensory neurons. Proteins were separated in the first dimension using nonequilibrium pH gradient electrophoresis (O’Farrell et al., 1977) and SDS slab gel electrophoresis in the second dimension (see Materials and Methods). A number of minor proteins released by sympathetic neurons are not visible here.

tinctly different in mobility from the majority of those released by cultures of neurons (Fig. 2d). Although some variation in the quantity, number, and types of proteins released by neurons occurred in different platings (particularly for sensory neurons); several proteins were found to be consistently and selectively released by either sympathetic or sensory neurons in each of 15 different platings made over a period of 1 year. Four proteins were consistently released by sympathetic neurons but not by sensory neurons or ganglionic nonneuronal cells (circled proteins in Fig. 2a; see also Table 1); whereas, two proteins were consistently released by sensory but not by sympathetic neurons or ganglionic nonneuronal cells (indicated by arrows in Figs. 2b, c; see also Table 1). Interestingly,

three of the proteins released by sensory neurons grown in serum-free medium had molecular weights and isoelectric points (140 kDa, p1 5.9; 111 kDa, p1 5.0; 75 kDa, p1 5.4) very similar to three proteins whose synthesis and/or release by sympathetic neurons is greatly increased by growing sympathetic neurons in heart cell conditioned medium (see Sweadner, 1981).

Release of a Calcium-Dependent Metalloprotease Sympathetic and Sensory Neurons

by

Under appropriate conditions, proteases released by neurons can be visualized as clear bands in darkly stained polyacrylamide gels containing gelatin. A 62kDa protease released by all three neuronal populations

RANDALL

PROTEINS

RELEASED

TABLE 1 SELECTIVELY BY CULTURES OR SENSORY NEURONS

N. PITTMAN

95

Proteases

OF SYMPATHETIC

MW (x10-3)

PI

SCG

170 115 82 82

5.4 5.3 5.5 4.9

+a + + +

-b

-

-

-

180 54

5.8 5.3

-

+ +

+ +

DRG

New-ma1

Trigeminal

Note. These represent the proteins that were consistently selectively released by either sympathetic or sensory neurons different platings over the period of 1 year. The isoelectric (p1 values) were obtained from two-dimensional gels in isoelectric focusing was used as the first dimension (O’Farrell, ’ + = released. b - = not detected.

62K-

and in 15 points which 1975).

Con

was visible when gels were incubated at neutral pH in the presence of l-5 mM calcium (Fig. 3). The band of proteolytic activity was not visible in the absence of calcium or in the presence of magnesium or manganese (Fig. 3), and very little activity was observed at calcium concentrations below 100 palm(not shown). Two additional calcium-dependent bands of proteolytic activity were present at 57 and 64 kDa. These two bands increased in intensity following several freeze-thaw cycles or after samples sat at room temperature for several hours. They also had an identical inhibitor profile to the 62-kDa protease. The calcium-dependent protease was not inhibited by N-ethylmaleamide, iodoacetate, or leupeptin (Fig. 4), and was therefore not a member of the wellcharacterized family of calcium-dependent thiol proteases (Meyer et ah, 1964; Guroff, 1964; Kishimoto et ah, 1981; Zimmerman and Schlaefer, 1982). Similarly ineffective were inhibitors of aspartate and serine proteases (Table 2). Treatment of released proteins with chelating agents prior to gel electrophoresis eliminated proteolytic activity (Table 2); therefore the 62kDa protein is a calcium-dependent metalloprotease. Several properties of this protease (Table 2) are consistent with its being a collagenase. Attempts to determine if collagenase activity was being released by sympathetic or sensory neurons using radiolabeled Type I collagen in conventional assays (Hu et al., 1978; Cawston and Murphy, 1981) for collagenase activity were negative. Because of the small quantities of protein released by the neurons and the possible presence of inhibitors of collagenase activity in the samples, a gel method was used to determine if collagenase activity was being released. If inhibitors were present,

Ca

Mg

Mn

FIG. 3. Calcium-dependent protease released by neurons. Identical samples of released proteins from trigeminal ganglion neurons were loaded into each lane of a gelatin-containing gel followed by SDS gel electrophoresis (see Materials and Methods). The lanes were then separated and incubated in 20 mM Tris-HCl, pH 7.3, without divalent cations (lane l), with 5 mM calcium (lane 2), with 5 mM magnesium (lane 3), or with 5 mM manganese (lane 4) for 15 hr followed by protein staining.

62K-

con

NEM

IA

Leu

FIG. 4. Inability of inhibitors of thiol proteases to inhibit the calcium-dependent protease. Identical samples of released proteins from trigeminal ganglion neurons were run as described in Fig. 3 and gel slices incubated in 20 mM Tris-HCl, 5 mM calcium, pH 7.3 (lane 1) containing 2 mM N-ethylmaleamide (lane 2), 3 mM iodoacetate (lane 3), or 0.2 mM leupeptin (lane 4).

96

DEVELOPMENTAL TABLE 2 METALLOPROTEASE RELEASED AND SENSORY NEURONS

CALCIUM-DEPENDENT

MW pH optimum Requirements Inhibited by Not inhibited

by

Distribution

Substrates

-

BIOLOGY

BY SYMPATHETIC

62,000 (proteins at 57,000 and 64,000 with identical inhibitor profiles) 7.0-7.5 mM Ca”; very little activity below 0.1 mM Ca’+ and no activity with mM Mgz+, Mn’+, or Sr2+ EDTA, EGTA 2-mercaptoethanol, N-ethylmaleamide, iodoacetate, pepstatin, pnitrophenylguanidinobenzoate, 6-aminocaproic acid, benzamidine, leupeptin, soybean trypsin inhibitor, egg white trypsin inhibitor, phosphoramidon, CBZ-Gly-Phe-amide, PMSF, DFP Released by neurons from sympathetic, dorsal root, and trigeminal ganglia; a small amount also appears to be associated with membrane fractions of these neurons Native and denatured Type I collagen but not casein, fibronectin, or albumin

then they should separate from the active protease during electrophoresis. When native [3H]collagen (Type I) was incorporated into gels and used as a substrate, high concentrations of chymotrypsin did not degrade

105K- * 6OK *

FIG. 5. Degradation of [3H]co11agen by a calcium-dependent metalloprotease released by sympathetic neurons. Native Type I [3H]collagen (0.3 &i; 600 ng) was crosslinked into a polyacrylamide gel and processed as described under Materials and Methods. Incubations were performed in 20 mM Tris-HC1 (pH 7.3) containing 5 mM calcium. Samples were: lane 2, 2 ng bacterial collagenase; lane 3, 500 ng chymotrypsin; lane 4, proteins released by sympathetic neurons treated with 2 mM EDTA prior to electrophoresis; lane 5, proteins released by sympathetic neurons; lane 1 contains MW markers (phosphorylase-b, 93 kDa; BSA, 68 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 30 kDa; and lysozyme 14 kDa). Arrows indicate activities of bacterial collagenase (105 kDa) and protease released by sympathetic neurons (60 kDa).

VOLUME

110, 1985

the collagen (Fig. 5 lane 3) whereas both bacterial collagenase and proteins released by neurons did (Fig. 5 lanes 2 and 5). Although native collagen was degraded by the released proteins, the proteolytic activity present in released proteins was much more active against gelatin than collagen. Proteolytic activity was detectable in [3H]gelatin gels at concentrations at least lOOOfold lower than that needed to detect activity in [3H]collagen gels (not shown). Nevertheless, a calciumdependent metalloprotease did degrade collagen (Fig. 5 lane 5) whereas it did not degrade fibronectin (Fig. 6 lane 4). Chymotrypsin degraded both gelatin (not shown) and fibronectin (Fig. 6 lanes 1 and 2) which indicated that its inactivity in collagen gels was due to the maintenance of the collagen structure within the gel and not due to inactivation of the enzyme by the gel procedure. The proteolytic activity associated with bacterial collagenase had a MW of approximately 105 kDa, consistent with the molecular weight for this enzyme, while the neuronal proteolytic activity had a MW of about 60 kDa suggesting that it may be associated with the calcium-dependent metalloprotease(s) described above. The neuronal protease was inhibited by treatment with EDTA prior to electrophoresis (Fig. 5 lane 4) and was dependent on the presence of added calcium (not shown). Release of a Urokinase-like Plasminogen Sympathetic and Sensory Neurcms

Activator

by

When plasminogen was included in polyacrylamide gels along with gelatin, two additional bands at 51 and

25K

1

2

3

4

FIG. 6. Inability of calcium-dependent metalloprotease to degrade [?]fibronectin. [‘251]Fibronectin (1.6 &i; 600 ng) was crosslinked into a polyacrylamide gel and processed as described under Materials and Methods. Incubations were performed in 20 mM Tris-HCl (pH 7.3) containing 5 mM calcium. Samples were: lane 1, 0.5 ng chymotrypsin; lane 2,5 ng chymotrypsin; lane 3,10 ng bacterial collagenase; lane 4, proteins released by sympathetic neurons.

RANDALL

N. PITTMAN

Neuronal

97

Proteases

51 K-

62K51K32K-

32K-

&UK

Con pH5

FGB

Con

ACA

Benz

Leu

FIG. 7. The plasminogen activator released by sympathetic neurons. Identical samples of released proteins from sympathetic neurons were run as described in Fig. 3 in a gelatin and plasminogencontaining gel (see Materials and Methods). Gel slices were incubated in 20 mM Tris-HCI, 5 mM calcium, pH 7.7 (con) containing 40 pM pnitrophenylguanidinobenzoate (PGB), 10 mM 6-aminocaproic acid (ACA), 2 mM benzamidine (Benz), 0.2 mM leupeptin (Leu), or at pH 5.0. The plasminogen-dependent proteolytic activity is present at 51 and 32 kDa. Note that none of the drugs inhibit the 62-kDa Cat+dependent metalloprotease.

32 kDa were observed (Fig. 7, Con). Since neither band was observed in gels without plasminogen, these two proteases are plasminogen activators. Fresh samples of released proteins from sympathetic or sensory neurons contained about equal amounts of the 51- and 32-kDa proteases, but allowing samples to sit at room temperature resulted in an increase in the activity at 32 kDa and a concomitant decrease in activity at 51 kDa. Such a decrease in molecular weight without loss of activity is characteristic of urokinase-like plasminogen activators, but not of other types of plasminogen activators. Decreases in plasminogen activator activity occurred following incubation of gel slices with inhibitors of plasminogen activator (benzamidine), plasmin (6-aminocaproic acid), or both (leupeptin and p-nitrophenylguanidinobenzoate; Fig. 7). None of these inhibitors affected the calcium-dependent metalloprotease (Fig. 7). Rabbit antiserum produced against human urokinase immunoprecipitated the plasminogen activator released by rat sympathetic neurons (Fig. 8). Control rabbit serum or antiserum against human plasminogen; however, did not precipitate any proteolytic activity. Physiochemical properties of the neuronal plasminogen activator are given in Table 3.

-PIas

a-UK

FIG. 8. Precipitation of neuronal plasminogen activator with rabbit antiserum against human urokinase. Immunoglobulins present in control rabbit serum (con), or rabbit antiserum against urokinase (UK), or plasminogen (Plas) were absorbed onto Sepharose-linked protein A. This was then treated with 1 mM DFP to inactivate serine proteases and washed in buffer (50 mM Tris, 0.15 M NaCl, 5 mM EDTA, 0.5% NP-40, pH 7.4) to remove unreacted DFP. Immune complexes were incubated with 50 ~1 (lanes 1 and 2) or 250 ~1 (lanes 3 and 4) of released proteins from sympathetic neurons followed by washing four times in buffer and incubation in 2% SDS to dissociate complexes. Samples were run in gelatin and plasminogen-containing gels (see Materials and Methods) and incubated for 14 hr at 30°C in 20 mM Tris-HCl, pH 8.3.

Sites of Protease Release Sympathetic neurons were plated at a high density in a confined area (5-mm diameter) in a 3%mm culture dish and allowed to grow for 5 weeks. Three Teflon rings with vacuum grease on the bottom were then placed into the dish to form a three-chambered dish (Fig. 9). The inner chamber contained cell bodies and

NEURONAL MW pH optimum Inhibited by Not inhibited Distribution

Other

by

characteristics

TABLE 3 PLASMINOGEN

ACTIVATOR

51,000-converted to an active 32,000 form if allowed to sit at RT 8.0-8.5 DFP, p-nitrophenylguanidinobenzoate, 6aminocaproic acid, benzamidine, leupeptin N-ethylmaleamide, EDTA, pepstatin Released by sympathetic, trigeminal, and DRG neurons; present in membrane fractions from these neurons The plasminogen activator released by neurons is precipitated by rabbit antiserum against human urokinase

98

DEVELOPMENTAL

FIG. 9. Three-chambered dish to determine sites area (5 mm) in a 35-mm culture dish and allowed form the three chambers so that cell bodies and chamber (c), and distal processes and growth cones

BIOLOGY

VOLUME

110, 1985

of protease releast ? (a). Sympathetic neurons were plated at a high density in a /confined to grow for 5 week .s. Three Teflon rings with vacuum grease on the bottom were used to dendrites were pr *esent in the central chamber (b), proximal processes in the middle in the outer than lber (d). X200 for (b-d); life size for (a).

dendrites, the middle chamber axonal processes, and the outer chamber, distal processes and growth cones (Fig. 9). Light microscopic visualization indicated that the inner and middle chambers contained practically all of the tissue mass present in the dish. Even though the amount of tissue in the outer chamber was exceedingly small, a large fraction of both the plasminogen activator and the metalloprotease was released into this chamber by distal processes and/or growth cones (Fig. 10). Quantitative measurement of plasminogen activator released by the different parts of the neuron is given in Table 4. DISCUSSION

Neuronal cultures free of nonneuronal cells provide an opportunity to identify and characterize proteins released by different types of neurons. Sympathetic

neurons from the superior cervical ganglion and sensory neurons from dorsal root and trigeminal ganglia all release 12-15 major proteins, most of which are different from those released by ganglionic nonneuronal cells (Fig. 2). Two proteins (180 kDa, p1 5.8 and 54 kDa, p1 5.3) were consistently found to be released by sensory neurons from dorsal root and trigeminal ganglia but not sympathetic neurons. These same two proteins are also released by sensory neurons from the nodose ganglion (preliminary observations). Thus, these proteins are released by neural crest- as well as placodederived sensory neurons, but not by sympathetic neurons. These proteins may serve as markers for sensory neurons from a variety of sources including cell lines. Sympathetic neurons from the superior cervical ganglion released four major proteins into the culture medium that were not released by the sensory neurons (Table 1). As with the “sensory specific” proteins, these

RANDALL

N. PITTMAN

62KFYI+

32K-

outer

Inner middle

FIG. 10. Release of proteases by different parts of the neuron. An equal portion (25%) of the total medium present in the outer chamber containing distal processes and growth cones (outer), the inner chamber containing cell bodies (inner), and the middle chamber containing proximal processes (middle) (see Fig. 9) was loaded and run in a gelatinand plasminogen-containing gel. The gel was processed as described in Fig. 3 and under Materials and Methods.

proteins may also serve as markers for assigning sympathetic function to cultures of primary or clonal cells. At present, specific functions cannot be assigned to most of the proteins released by these neurons. However, enzymatic activities are associated with two released proteins. One is a calcium-dependent metalloprotease, and the other a plasminogen activator. The calcium-dependent protease is not a member of the well-characterized calcium-dependent thiol proteases found in the brain (Guroff, 1964, Schlaepfer and Freeman, 1980; Kishimoto et ab, 1981; Zimmerman and Schlaepfer, 1982; Malik et ah, 1984), nor is its activity decreased by inhibitors (phosphoramidon and CBZGly-Phe-amide) of the calcium-dependent metalloprotease that may be involved in neurotransmission (Baxter et al., 1983). Its requirement of millimolar calcium for maximum activity suggests that this protease has little activity while inside the neuron, but would become active immediately upon release. However, its substrate profile would indicate that it probably has little general proteolytic activity even outside the neuron. It had very little activity against albumin, casein, or fibronectin whereas it could degrade both native and denatured collagen (Type I) incorporated into gels. Denatured collagen (gelatin) was a much better substrate than was native collagen which may indicate that this

Neuronal

99

Proteases

enzyme is a gelatinase. Its physiochemical properties are similar to those reported for tissue gelatinase (Sellers et al., 1978, Vaes et al., 1978; Sakyo et al., 1983; Galloway et ah, 1983). It is also possible that the activity represents two proteases with very similar properties (both calcium-dependent metalloproteases with approximately the same molecular weights), one a collagenase and the other a gelatinase. Fibroblasts release a collagenase/gelatinase pair with almost identical properties (Vater et ah, 1983; Aggeler et al., 1984). In some cases the gelatinase serves to activate coreleased procollagenases (Vater et al., 1983) and in other cases it has proteoglycanase activity (Werb et al., 1978; Frisch et ccl., 1983; Galloway et al., 1983). Thus it is possible that the neuronal growth cones release both a collagenase and a gelatinase. The release of such enzymes from the distal processes of developing neurons could be useful in neurite outgrowth through collagencontaining extracellular matrices. Future work will be directed at determining whether other extracellular matrix components such as proteoglycans are also substrates for the calcium-dependent metalloprotease(s). Plasminogen activator activity has been suggested to be involved in granule cell migration in the cerebellum (Krystosek and Seeds, 1981b; Moonen et al., 1982; Soreq and Miskin, 1983). The results presented here and those of Krystosek and Seeds (1984) indicate that neurons can spontaneously release plasminogen activator. The present characterization further shows a physiochemical and immunological similarity between the neuronal plasminogen activator and urokinase. Whereas the properties of the calcium-dependent metalloprotease suggest that its physiological substrate may be a component of the extracellular matrix, the substrate for the neuronal plasminogen activator is less certain. Although plasminogen is the normal substrate for plasminogen activators, it is not clear that

AMOUNT

Chamber Somas/dendrites Proximal processes Distal processes/ growth cones

TABLE 4 OF PLASMINOGEN ACTIVATOR SYMPATHETIC NEURONS

munits

PA/chamber/24

RELEASED

hr

BY

% of total PA in each chamber

3.37 + 0.26 0.80 + 0.12

51 12

2.48 If: 0.19

37

Note. Data represent the mean f SEM of four cultures similar to the one in Fig. 9 containing about 1000 neurons each. Plasminogen activator activity was determined using an [‘261]fibrin plate assay (Materials and Methods).

100

DEVELOPMENTAL

BIOLOGY

this serum protein is available to developing neurons. If plasminogen were in the extracellular space and accessible to neurons, it could serve as a reservoir of general proteolytic activity available to cells releasing plasminogen activator. Substrates other than plasminogen may also exist. Quigley (1979) has shown that morphological changes occur in chick embryo fibroblasts following exposure to highly purified urokinase in the absence of plasminogen. This study suggested that the surface of these cells may contain a substrate for plasminogen activator. Keski-Oja and Vaheri (1982) found that a 66-kDa protein in the pericellular matrix of fibroblasts appeared to be a substrate for urokinase. Therefore, even if plasminogen is not available to developing neurons, specific substrates for plasminogen activator may exist in the extracellular matrix or on the surface of certain cells. Neither the calcium-dependent metalloprotease nor the plasminogen activator was released in large quantities by the proximal portions of neurites (middle chamber, Fig. 10). Because the chamber containing these neuronal processes also contained a large fraction of the varicosities (sites of transmitter release), it would seem likely that neither of the proteases would be involved in synaptic transmission. Consistent with this idea are experiments showing that depolarizing neurons with 20 mM potassium had no effect on the release of these proteases (unpublished observations). The largest amount of proteases was released into the inner chamber (Fig. 10) which contained the bulk of the neuronal tissue including cell bodies, dendrites, and any synapses present. It may be that dendrites release these proteases or that the proteases are released by varicosities which form synapses but not by “free” varicosities such as those present in the middle chamber. Histological studies using neuroblastoma cells and sensory neurons have indicated that plasminogen activator is released from cell bodies (Krystosek and Seeds, 1981a, 1984; Soreq et al., 1983); therefore it may be that the neuronal somas are releasing proteases into the inner chamber. Of particular significance, however, is that almost as much proteolytic activity was released into the outer chamber containing distal processes and growth cones as in the inner chamber (Fig. 10; Table 3) even though the amount of tissue in the outer chamber was several orders of magnitude less. Thus, the “specific activity” of released proteases was much greater in the chamber containing distal processes and growth cones. Because both the calciumdependent metalloprotease and the plasminogen activator are released by distal processes and/or growth cones, both proteases could be involved in growth cone function. The physiological role of these proteases is unknown, although interactions with the local environ-

VOLUME

110.1985

ment including the extracellular matrix and target cells are likely possibilities. Their functional roles may depend not only on available substrates, but also on diffusible and surface-bound protease inhibitors known to be present in the local environment of the neuron (Pittman, 1984). This work was supported by grants to Paul H. Patterson from the NINCDS, the McKnight Foundation, and The Rita Allen Foundation, and by an MDA postdoctoral fellowship to R.P. I thank Doreen McDowell for preparation of cell culture media and Allison Doupe and Eve Wolinsky for NGF. Special thanks go to Paul Patterson in whose laboratory this work was performed for providing a moving environment and many stimulating beer hours. REFERENCES AGGELER, J., FRISCH, S. M., and WERB, Z. (1984). Collagenase is a major gene product of induced rabbit synovial fibroblasts. J. Cell BioL 98,1656-1661. BARDE, Y.-A., EDGAR, D., and THOENEN, H. (1983). New neurotrophic factors. Annu. Rev. PhysioL 45, 601-612. BAXTER, D. A., JOHNSTON, D., and STRITTMATTER, W. J. (1983). Protease inhibitors implicate metalloendoprotease in synaptic transmission at the mammalian neuromuscular junction. Proc. NatL Acad. Sci. USA 80,4174-4178. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. B&hem 46, 83-88. BOTTENSTEIN, J. E., SKAPER, S. D., VARON, S. S., and SATO, G. H. (1980). Selective survival of neurons from chick embryo sensory ganglionic dissociates utilizing serum-free supplemented medium. Exp. Cell Res. 125, 183-190. BRADSHAW, R. A. (1978). Nerve growth factor. Annu. Rev. Biochem. 47, 191-216. CAWSTON, T. E., and MURPHY, G. (1981). Mammalian collagenases. In “Methods in Enzymology” (L. Lorand, ed.), Vol. 80, pp. 711-722. Academic Press, New York. DEUTSCH, D. G., and MERTZ, E. T. (1970). Plasminogen: Purification from human plasma by affinity chromatography. Science (Washington, D. C.) 170, 1095-1096. EDELMAN, G. M. (1983). Cell adhesion molecules. Science (Washington, D. C.) 219,450-457. FRISCH, S. M., CHIN, J. R., and WERB, Z. (1983). Molecular cloning of a cDNA encoding a secreted connective tissue-degrading metalloproteinase induced by changes in cytoskeletal structure. J. Cell BioL 97, 403a. GALLOWAY, W. A., MURPHY, G., SANDY, J. D., GAVRILOVIC, J., CAWSTON, T. E., and REYNOLDS, J. J. (1983). Purification and characterization of a rabbit bone metalloproteinase that degrades proteoglycan and other connective tissue components. Biochem J. 209, 741-752. GUROFF, G. (1964). A neutral calcium-activated proteinase from the soluble fraction of rat brain. J. BioL Chem 239, 149-155. HAWROT, E., and PATTERSON, P. H. (1979). Long-term culture of dissociated sympathetic neurons. In “Methods in Enzymology” (W. B. Jakoby and I. H. Pastan, eds.), Vol. 58, pp. 574-584. Academic Press, New York. HEUSSEN, C., and DOWDLE, E. B. (1980). Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem 102, 196-202. Hu, C.-L., CROMBIE, G., and FRANZBLAU, C. (1978). A new assay for collagenolytic activity. AnaL Biochem 88, 638-643.

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