Journal 01Neurochemislry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry
Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastorna Cells *?Thomas B. Shea, *Mary Lou Beermann, and *$§Ralph A. Nixon *Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, and Departments of ?Biological Chemistry and Molecular Pharmacology and $Psychiatry and $Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, U.S.A.
Abstract: Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucylleucyl-norleucinal (CI; preferential inhibitor of micromolar M concalpain but also inhibits millimolar calpain) at siderably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucylleucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable
neuntes in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain. Key Words: Neudifferentiation-Neuroblastomaritogenesis-Neuronal NB2a/dl-Protease-Protease inhibitors-calpainThrombin. Shea T. B. et al. Multiple proteases regulate neurite outgrowth in NB2a/dl neuroblastoma cells. J. Neurochem. 56, 842-85 I (199I).
Proteolysis is instrumental at several stages during neuronal differentiation and neurite outgrowth (for review, see Monard, 1985). Specific proteases are required to allow migrating neuroblasts to arrive at their final destination (Becherer and Wachsman, 1980 Krystosek and Seeds, 1978, 1981a; Moonen et al., 1982; Soreq and Miskin, 1983; Valinsky and LeDouarin, 1985; Grossman et al., 1987). Neurons release proteases (Krystosek and Seeds, 198 1 a, 1984; Soreq and Miskin, 1983; Alvarez-Buylla and Valinsky, 1985; Pittman, 1985), certain of which regulate the turnover of specific membrane proteins, and the inhibition of these proteases suppresses neuroblast migration and fosters the establishment of minor neuritic processes (Becherer and Wachsman, 1980; Gibson et al., 1984; Shea et al., 1985; Saito and Kawashima, 1988; Pittman et al., 1989; Sargent, 1989; Smalheiser, 1989a,b). Following neurite induction, a collagen/extracellular matrix-penetrating
proteolytic activity is localized along distal neurite areas and growth cones (Krystosek and Seeds, 198 1 b; Tosney and Landmesser, 1984; Pittman, 1985; Pittman and Williams, 1988; Pittman et al., 1989). Glial cells secrete proteins that promote neurite formation and possess potent inhibitory activity against tissue plasminogen activator, urokinase, and thrombin (Monard et al., 1983; Guenther et al., 1985; Monard, 1985; Stone et al., 1987). One of these proteins has recently been identified as a member of the cell-derived protease inhibitors termed “nexins” (Gloor et al., 1986). Localized application of protease inhibitors can chemotropically influence the direction of neurite outgrowth (Hawkins and Seeds, 1989). Furthermore, secreted protease inhibitors can bind to the extracellular matrix, and this binding increases their inhibitory activity and alters their specificity (Farrell et al., 1988). These results indicate that, in addition to inducing
~
Received May 22, 1990; revised manuscript received August 22, 1990; accepted August 23, 1990. Address correspondence and reprint requests to Dr. T. B. Shea at Ralph Lowell Laboratories, McLean Hospital, Belmont. MA 02 178, USA.
,4 bbreviafionsused: CI, N-acetyl-leucyl-leucyl-norleucinal; CII, Nacetyl-leucyl-leucyl-methional;SD, soma1 diameter.
842
MULTIPLE PROTEASES REGULATE NEURITE OUTGRO W T f f neuritogenesis, the surrounding glial environment can promote correct axonal pathfinding and target innervation. Thrombin, or a thrombin-like enzyme, present on the outer plasma membrane surface or as a serum component has been implicated in the regulation of neurite outgrowth since the specific thrombin inhibitor hirudin induces neuritogenesis as effectively as serum depletion (Gurwitz and Cunningham, 1988; Cunningham and Gurwitz, 1989). Conversely, hirudin was ineffective in studies where the synthetic leupeptin analogue ALLNal enhanced nerve growth factor-mediated neuritogenesis in PC12 cells, which led to the suggestion that calpain, and not thrombin, mediated this effect (Saito and Kawashima, 1988). Interestingly, ALLNal induced neuritogenesis only when nerve growth factor was present in the medium. This finding raises the possibility that the induction of neuritogenesis may not be equivalent to the enhancement of outgrowth of existing neurites, but instead may reflect separate events that are regulated by distinct proteases and protease inhibitors. In the present study, we examined this possibility by treating NB2a/dl cells with protease inhibitors. We previously showed that mouse NB2a/dl neuroblastoma cells rapidly elaborate neurites after serum is removed from the culture medium (Shea et al., 1985), suggesting the presence of a serum factor(s) that may suppress neuritogenesis. We observed in this study that multiple classes of protease inhibitors can influence neuritogenesis. One class of inhibitors induces the de novo outgrowth of unstable neurites in the presence of serum. Another class cannot initiate neuritogenesis, but does enhance the length of neurites elaborated from serumdeprived cells. We further demonstrate that these two classes of inhibitors exert complementary effects and together induce the outgrowth of stable neurites in the presence of serum. Portions of this research have been presented in abstract form (Shea et al., 1990~).
MATERIALS AND METHODS NB2a/dl cells (Shea et al., 1985, 1988) were plated at a density of lo5cells/ml in Dulbecco’s modified Eagle medium containing 10%horse serum and 25 pg/ml gentamycin (Sigma Chemical Co., St. Louis, MO, U.S.A.) in noncoated 35-mm2 petri plates or six-well trays (Flow Labs, McLean, VA, U.S.A.) in a humidified atmosphere of 95%air and 5% CO1. Primary astrocytic cultures (generous gift of Dr. Itzhak Fischer, E.K. Shriver Center, Waltham, MA, U.S.A.) prepared from neonatal rat brain as described (Fischer et al., 1990) were cultured in the same medium except that fetal bovine serum (Sigma) was substituted for horse serum. Twenty-four hours later, the medium was replaced with medium containing either 10 or 0.8% serum or deprived of serum to induce the elaboration of axonal neurites (Schubert et al., 1969, 1974; Seeds et al., 1970; Bottenstein and Sato, 1979; Shea et al., 1985). At this time cultures also received one or more of the following: the cysteine protease inhibitors leupeptin, N-acetyl-leucyl-leucylnorleucinal (CI), and N-acetyl-leucyl-leucyl-methional(CII; preferential inhibitors of micro- and millimolar calpain, re-
843
spectively; however, each inhibits both forms of calpain) added at 1-100 p M , soybean trypsin inhibitor, pepstatin, chymostatin, and aprotinin at 0.5-100 &ml; hirudin at 5 U/ml; thrombin at 0.1-100 pg/ml; colchicine at M ; cycloheximide at 0.5 pg/ml; dibutyryl cyclic AMP at 1 mM. All chemicals were purchased from Sigma except CI and CII, which were purchased from Boehringer-Mannheim (Indianapolis, IN, U.S.A.). Calpain was purified 950-fold from mouse brain by sequential chromatography on DE-52 cellulose, phenyl-Sepharose, and Ultrogel-AcA-44 as previously described (Nixon et al., 1986). Calpain was used at a final concentration of I pg/ml in cell-free assays and was added to culture medium at final concentrations of 10 and 100 pg/ ml. It should be noted that the final concentrations of proteases and inhibitors as reported above were based upon cellfree analyses and that the effective concentrations in culture medium may be substantially less than these values. At 4 or 24 h after the above treatments, duplicate cultures were rinsed in Tns-buffered saline (pH 7.4), fixed for 10 min in 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.2) at room temperature for 15 min, and then rinsed and stored in Tris-buffered saline. All experiments were camed out at least twice. In each experiment, between 100 and 200 cells in each of the fixed cultures were examined by phase-contrast microscopy. The relative extent of neurite outgrowth was quantitated by comparison of neurite length with respective soma1 diameter (SD) as previously described (Shea et al., 1985). All cells examined were grouped as follows: cells possessing neurites that were 4 SD. In some experiments, cells lacking detectable neurites were scored separately from those possessing neurites of < 1 SD. Each graphed value represents the average of the counts obtained from sets of duplicate cultures from at least two experiments (therefore, a total of at least four separate determinations). The effect of protease inhibitors on neuritogenesis was also examined by Student’s t test as follows. For analyses of the effect of hirudin on neurite induction, the ratio of numbers of cells with 2 1 SD neurites to the number of cells with < I SD neurites for 100-200 total cells in each of five cultures containing 0.8% serum plus hirudin (n = 5 ) was compared with that obtained for 100-200 total cells in each of eight cultures containing serum alone (n = 8). For analyses of the effect of leupeptin and CI on neurite length in serumdeprived cells, cells incubated for 24 h in the absence of serum and with and without protease inhibitors were scored simply as possessing one or more neurites of either
Multiple Proteases Regulate Neurite Outgrowth in NB2a/dl Neuroblastorna Cells *?Thomas B. Shea, *Mary Lou Beermann, and *$§Ralph A. Nixon *Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, and Departments of ?Biological Chemistry and Molecular Pharmacology and $Psychiatry and $Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, U.S.A.
Abstract: Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucylleucyl-norleucinal (CI; preferential inhibitor of micromolar M concalpain but also inhibits millimolar calpain) at siderably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucylleucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable
neuntes in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain. Key Words: Neudifferentiation-Neuroblastomaritogenesis-Neuronal NB2a/dl-Protease-Protease inhibitors-calpainThrombin. Shea T. B. et al. Multiple proteases regulate neurite outgrowth in NB2a/dl neuroblastoma cells. J. Neurochem. 56, 842-85 I (199I).
Proteolysis is instrumental at several stages during neuronal differentiation and neurite outgrowth (for review, see Monard, 1985). Specific proteases are required to allow migrating neuroblasts to arrive at their final destination (Becherer and Wachsman, 1980 Krystosek and Seeds, 1978, 1981a; Moonen et al., 1982; Soreq and Miskin, 1983; Valinsky and LeDouarin, 1985; Grossman et al., 1987). Neurons release proteases (Krystosek and Seeds, 198 1 a, 1984; Soreq and Miskin, 1983; Alvarez-Buylla and Valinsky, 1985; Pittman, 1985), certain of which regulate the turnover of specific membrane proteins, and the inhibition of these proteases suppresses neuroblast migration and fosters the establishment of minor neuritic processes (Becherer and Wachsman, 1980; Gibson et al., 1984; Shea et al., 1985; Saito and Kawashima, 1988; Pittman et al., 1989; Sargent, 1989; Smalheiser, 1989a,b). Following neurite induction, a collagen/extracellular matrix-penetrating
proteolytic activity is localized along distal neurite areas and growth cones (Krystosek and Seeds, 198 1 b; Tosney and Landmesser, 1984; Pittman, 1985; Pittman and Williams, 1988; Pittman et al., 1989). Glial cells secrete proteins that promote neurite formation and possess potent inhibitory activity against tissue plasminogen activator, urokinase, and thrombin (Monard et al., 1983; Guenther et al., 1985; Monard, 1985; Stone et al., 1987). One of these proteins has recently been identified as a member of the cell-derived protease inhibitors termed “nexins” (Gloor et al., 1986). Localized application of protease inhibitors can chemotropically influence the direction of neurite outgrowth (Hawkins and Seeds, 1989). Furthermore, secreted protease inhibitors can bind to the extracellular matrix, and this binding increases their inhibitory activity and alters their specificity (Farrell et al., 1988). These results indicate that, in addition to inducing
~
Received May 22, 1990; revised manuscript received August 22, 1990; accepted August 23, 1990. Address correspondence and reprint requests to Dr. T. B. Shea at Ralph Lowell Laboratories, McLean Hospital, Belmont. MA 02 178, USA.
,4 bbreviafionsused: CI, N-acetyl-leucyl-leucyl-norleucinal; CII, Nacetyl-leucyl-leucyl-methional;SD, soma1 diameter.
842
MULTIPLE PROTEASES REGULATE NEURITE OUTGRO W T f f neuritogenesis, the surrounding glial environment can promote correct axonal pathfinding and target innervation. Thrombin, or a thrombin-like enzyme, present on the outer plasma membrane surface or as a serum component has been implicated in the regulation of neurite outgrowth since the specific thrombin inhibitor hirudin induces neuritogenesis as effectively as serum depletion (Gurwitz and Cunningham, 1988; Cunningham and Gurwitz, 1989). Conversely, hirudin was ineffective in studies where the synthetic leupeptin analogue ALLNal enhanced nerve growth factor-mediated neuritogenesis in PC12 cells, which led to the suggestion that calpain, and not thrombin, mediated this effect (Saito and Kawashima, 1988). Interestingly, ALLNal induced neuritogenesis only when nerve growth factor was present in the medium. This finding raises the possibility that the induction of neuritogenesis may not be equivalent to the enhancement of outgrowth of existing neurites, but instead may reflect separate events that are regulated by distinct proteases and protease inhibitors. In the present study, we examined this possibility by treating NB2a/dl cells with protease inhibitors. We previously showed that mouse NB2a/dl neuroblastoma cells rapidly elaborate neurites after serum is removed from the culture medium (Shea et al., 1985), suggesting the presence of a serum factor(s) that may suppress neuritogenesis. We observed in this study that multiple classes of protease inhibitors can influence neuritogenesis. One class of inhibitors induces the de novo outgrowth of unstable neurites in the presence of serum. Another class cannot initiate neuritogenesis, but does enhance the length of neurites elaborated from serumdeprived cells. We further demonstrate that these two classes of inhibitors exert complementary effects and together induce the outgrowth of stable neurites in the presence of serum. Portions of this research have been presented in abstract form (Shea et al., 1990~).
MATERIALS AND METHODS NB2a/dl cells (Shea et al., 1985, 1988) were plated at a density of lo5cells/ml in Dulbecco’s modified Eagle medium containing 10%horse serum and 25 pg/ml gentamycin (Sigma Chemical Co., St. Louis, MO, U.S.A.) in noncoated 35-mm2 petri plates or six-well trays (Flow Labs, McLean, VA, U.S.A.) in a humidified atmosphere of 95%air and 5% CO1. Primary astrocytic cultures (generous gift of Dr. Itzhak Fischer, E.K. Shriver Center, Waltham, MA, U.S.A.) prepared from neonatal rat brain as described (Fischer et al., 1990) were cultured in the same medium except that fetal bovine serum (Sigma) was substituted for horse serum. Twenty-four hours later, the medium was replaced with medium containing either 10 or 0.8% serum or deprived of serum to induce the elaboration of axonal neurites (Schubert et al., 1969, 1974; Seeds et al., 1970; Bottenstein and Sato, 1979; Shea et al., 1985). At this time cultures also received one or more of the following: the cysteine protease inhibitors leupeptin, N-acetyl-leucyl-leucylnorleucinal (CI), and N-acetyl-leucyl-leucyl-methional(CII; preferential inhibitors of micro- and millimolar calpain, re-
843
spectively; however, each inhibits both forms of calpain) added at 1-100 p M , soybean trypsin inhibitor, pepstatin, chymostatin, and aprotinin at 0.5-100 &ml; hirudin at 5 U/ml; thrombin at 0.1-100 pg/ml; colchicine at M ; cycloheximide at 0.5 pg/ml; dibutyryl cyclic AMP at 1 mM. All chemicals were purchased from Sigma except CI and CII, which were purchased from Boehringer-Mannheim (Indianapolis, IN, U.S.A.). Calpain was purified 950-fold from mouse brain by sequential chromatography on DE-52 cellulose, phenyl-Sepharose, and Ultrogel-AcA-44 as previously described (Nixon et al., 1986). Calpain was used at a final concentration of I pg/ml in cell-free assays and was added to culture medium at final concentrations of 10 and 100 pg/ ml. It should be noted that the final concentrations of proteases and inhibitors as reported above were based upon cellfree analyses and that the effective concentrations in culture medium may be substantially less than these values. At 4 or 24 h after the above treatments, duplicate cultures were rinsed in Tns-buffered saline (pH 7.4), fixed for 10 min in 4% paraformaldehyde in 0.1 Mphosphate buffer (pH 7.2) at room temperature for 15 min, and then rinsed and stored in Tris-buffered saline. All experiments were camed out at least twice. In each experiment, between 100 and 200 cells in each of the fixed cultures were examined by phase-contrast microscopy. The relative extent of neurite outgrowth was quantitated by comparison of neurite length with respective soma1 diameter (SD) as previously described (Shea et al., 1985). All cells examined were grouped as follows: cells possessing neurites that were 4 SD. In some experiments, cells lacking detectable neurites were scored separately from those possessing neurites of < 1 SD. Each graphed value represents the average of the counts obtained from sets of duplicate cultures from at least two experiments (therefore, a total of at least four separate determinations). The effect of protease inhibitors on neuritogenesis was also examined by Student’s t test as follows. For analyses of the effect of hirudin on neurite induction, the ratio of numbers of cells with 2 1 SD neurites to the number of cells with < I SD neurites for 100-200 total cells in each of five cultures containing 0.8% serum plus hirudin (n = 5 ) was compared with that obtained for 100-200 total cells in each of eight cultures containing serum alone (n = 8). For analyses of the effect of leupeptin and CI on neurite length in serumdeprived cells, cells incubated for 24 h in the absence of serum and with and without protease inhibitors were scored simply as possessing one or more neurites of either
