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Staurosporine-induced morphological differentiation of human neuroblastoma cells
CelJ Biology
International
Reports,
Vol. 75, No. 2, February
STAUROSPORINE-INDUCED DIFFERENTIATION OF HUMAN Thomas
MORPHOLOGICAL NEUROBLASTOMA
B. Shea1>2 and Mary
161
1991
CELLS
Lou Beermann
‘Ralph Lowell Laboratories, McLean Hospital, Belmont, MA 02178 2Depaxtment of Psychiatry. Harvard Medical School. Boston, MA 0211.5
ABSTRACT SH-SY-SY human neuroblastoma cells rapidly elaborated an extensive network ofneuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, II7 ] l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of CAMP- and cGMP-dependent protein kinases but not protein kinasc C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly. suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement ot existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.
INTRODUCTION Neuroblastoma cells undergo morphological and biochemical differentiation following treatment with a varitey of agents inducing compounds that raise CAMP levels (Prasad and Hsie, 1971; Shea et al., 1985, 1988), retinoic acid (Fischer et al., 1986; Pahlman et al., 1984; Shea et al., 1985; Sidell et al., 1983) serum deprival (Bottenstein and Sato, 1979; Seeds et al., 1970; Schubert et al., 1969, 1974; Shea et al.. 1985, 1990), certain protease inhibitors (Becherer and Wachsman, 1980; Cunningham and Gurwitz, 1989; Gibson et al., 1984; Gurwitz and Cunningham, 1988; Pittman et al., 1989; Saito and Kawashima, 1988; Sargent, 1989; Shea et al., 1990; Smalheiser, 1989a,b) increases in extracellular (Reboulleau, 1986) or intracellular (Anglister et al., 1982; Duprat and Kan, 1981; Gurevitch et al., 1982; Shea, 1990) calcium levels, gangliosides (Leskawa and Hogan, 1985; Roisen et al., 1981; Tsuji et al., 1983), and inhibitors of protein kinase C (Felipo et al., 1990; Hashimoto et al., 1989; Heikkila et al., 1989; Minana et al., 1990; Tsuda et al., 1989). By virtue of their ability to express many properties of differentiating and mature neurons following these treatments, neuroblastoma cells represent powerful model systems for studying the sequence of events underlying and accompanying the outgrowth and stabilization of neurites. The human neuroblastoma cell line, SH-SY-5Y has been reported to undergo morphological differentiation following treatment with the protein kinase C inhibitor H7 (Heikkila et al., 1989). Since H7 inhibits CAMP-dependent and cGMP-dependent kinases in addition to protein kinase C (Tsuji et al., 1983), however, we attempted to clarify the respective roles of these protein kinases in neuritogenesis. To accomplish this, we examined the effects on morphological differentiation of these cells following treatment with another protein kinase C inhibitor, staurosporine (Kase et al., 1987; Nakanishi et al., 1988; Ozaki et al.,
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1987; Tamaokli et al., 1986), and HA1004, which inhibits CAMP-dependent and cGMP-dependent kinases but not protein kinase C (Hidaka et al., 1984). We observed that staurosporine and H7 induced morphologically distinct.neurites. but that HA1004 did not, confirming earlier suggestions that protein kinase C is involved in the regulation of neurite outgrowth. MATERIALS AND METHODS SH-SY-SY cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (FCS) at 370C in a humidified atmosphere containing 10% C02. Twenty-four hrs after plating, the cultures were treated with one or more of the following : staurosporine ( I-lOOpM), H7 (lo-500pM), HA-1004 (ZOOyM), colchicine ( IpM), and cycloheximide (OSpg/ml) for 6 and 24hrs. In some experiments, cultures treated for 6hrs were washed, the medium replaced with fresh medium lacking test compound(s), and incubation continued for a total of 24hrs. Stock solutions of inhibitors were prepared in 100% DMSO, then diluted in culture medium so that the tinal concentration of DMSO did not exceed 0.1%; this concentration did not induce morphological differentiation by 6hr, but did induce the outgrowth of short neurites by 24hrs of continuous treatment. Immediately after incubation, cultures were rinsed with phosphate-buffered saline (PBS: pH 7.4), fixed for ISmin at room temperature with 4% paraformaldehyde in O.lM phosphate buffer (pH 7.4), returned to PBS. then photographed and quantitated by phase-contrast microscopy (Shea et al., 1985). SH-SY-SY cells (originally obtained from the stocks of Dr. June L. Bielder, Memorial Sloan-Kettering Cancer Center, Rye, New York) were provided by Dr. Itzhak Fischer, E.K. Shriver Center, Waltham, MA, USA. Kinase inhibitors were generous gifts of Dr. Ubaldo Leli, McLean Hospital, Belmont MA, USA. All other reagents and chemicals were obtained from Sigma Chem. Co.(St. Louis, MO). RESULTS SH-SY-SY cells posses only short filopodia-like neurites; however, these cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine (Fig. I). Despite this extensive outgrowth, these neurites were retracted within 24hr following removal of inhibitor. As previously reported, another inhibitor of protein kinase C, H7, also induced rapid neurite outgrowth (Heikkila et al., 1989; Hidaka et al., 1984). This outgrowth was also reversible following the removal of this compound. Although both inhibitors induced neurite outgrowth, staurosporine-treated cells elaborated a branching network of neurites, in addition to longer, unbranching neurites, while H7-treated cells elaborated mostly long,unbranching neurites, and lacked a similar network of shorter neurites. Quantitative analysis of this induction of differing neurite morphologies revealed that 85.4+15.1% of cells in staurosporine-treated cultures possessed a neuritic network along with one or more long, unbranching neurites, while only 11.3+7.9% of cells in H-7-treated cultures elaborated a network in addition to long unbranching neurites (p< 0.0005: Student’s t test).
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Figure 1: Induction of morphological differentiation of human neuroblastoma cells by inhibitors of protein kinase C. Phase-contrast micrographs of fixed SH-SY-SY cells cultured under the following conditions: Panel A, no additional additives for 24hrs ; Panel B, IpM s taurosporine for 6hrs. Arrows indicate the elaborate network of branching neurites near cell bodies; panel C, 1pM staurosporine for 24hrs; panel D, 1pM staurosporine for 6hrs followed by fresh medium without staurosporine for 20hrs. Note the loss of neurites; Panel E, 1pM H7 for 6hrs; panel F, 1pM H7 for 24hrs, arrows indicate a long, unbranching neurite. Note the lack of a neuritic network surrounding cell bodies; panel G, 1uM H7 for 4hrs followed by fresh medium without H7 for 20hrs. Note the loss of net&es; Panel H, 1OOpM HA-1004 for 6hrs. Neurites are not observed, panel C, 1OOpMHA-1004 for 24hrs. Note the presence of only short neurites; panel D, solvent (DMSO) alone for 24hrs. Note that short neurites are induced by treatment for 24hr with DMSO.
In contrast to the above results, HA-1004, which inhibits CAMP- and cGMP-dependent protein kinases but not protein kinase C (Hidaka et al., 1984), did not induce neuritogenesis over that observed for the solvent control (i.e., DMSO alone; see also Kimhi et al., 1976) when added at concentrations of up to 200 times those e&ctive for staurosporine and H7.
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Since treatment with protein kinase C inhibitors did not induce terminal differentiation of these cells, we examined whether or not these treatments induced alterations in genetic expression, or rather, the reorganization of existing materials. This was accomplished by protein syntheses inhibition by treatment with cycloheximide, and by prevention of microtubule polymerization by colchicine, during treatment with staurosporine (Fig. 2). Neurite outgrowth was unaffected by cycloheximide, indicating its independence of protein synthesis, but was blocked by colchicine, indicating the requirement for microtubule assembly as a driving force.
Firure 2: Neurites induced by protein kinase C inhibition are dependent upon microtubule assembly but independent of protein synthesis. Phase-contrast micrographs of fixed SH-SY-SY cells cultured under the following conditions: panel A, staurosporine for 6hrs; Panel B, cycloheximide for 90min followed by staurosporine and cycloheximide for 6 additional hrs; panel C, staurosporine and colchicine for 6hrs. Note that the staurosporine-induced elaboration 01 neurites is not inhibited by cyclohexime but is prevented by colchicine.
DISCUSSION Two inhibitors of protein kinase C, staurosporine and H7 induced the outgrowth of morphologically distinct neurites. Staurosporine-treated cells elaborated a branching network of neurites, in addition to longer, unbranching neurites, while H7-treated cells elaborated mostly long, unbranching neurites , and lacked a similar network of shorter neurites. These morphological differences may reflect the respective inhibition of different protein kinase C subtypes; although seven distinct subtypes of protein kinase C exist (Nishizuka, 1989). H7 selectively inhibits the alpha subtype (Pelosin et al., 1990), and the particular subtype(s) inhibited by staurosporine, unknown at present, may differ. The independence of staurosporine-mediated neurite outgrowth on protein synthesis, coupled with its requirement for microtubule assembly, indicates that
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the outgrowth of neurites induced by inhibition of protein kinase C is apparently mediated by the polymerization of cytoskeletal precursors or by a reorganization of existing cytoskeletal structures. A similar independence of the need for additional synthesis of RNA or protein has been reported for H7-mediated induction of neurites in N18TG2 neuroblastoma and cultured cerebellar cells (Tsuda et al., 1989). The data of the present study, and that of Tsuda and colleagues imply that specific phosphorylation events are sufficient to restrict the outgrowth of neurites. lnteres tingly. activator of protein kinase C, phorbol ester 12-Otetracedanoylphorbol-13-acetate (TPA) has previously been reported to induce limited outgrowth of neurites from SH-SY-SY cells (Akerman et al., 1984: Pahhnan et al., 1981; Spinelli et al., 1982: Heikkila et al., 1989) and other neuronal cell lines (Hsu et al., 1989). An explanation for the apparent discrepancy of how both inhibitors and activators of protein kinase C can induce neuritogenesis lies in the observation that, upon activation by TPA, protein kinase C associates with the plasma membrane, resulting in a transient downregulation of enzyme activity (Ballester and Rosen, 1985: Blackshear et al.. 1985: Krafi and Anderson, 1983). This line of reasoning is strengthened by the observation that mutant lines, including the human neuroblastoma IMR 33 (Jalava et al.. 1988). in which protein kinase C does not associate with the membrane following TPA-treatment do not exhibit morphological differentiation in the presence of TPA (Homma et al.. 1986). TPA-induced outgrowth ti-01x1 SK SY-SY cells was enhanced by the simultaneous addition of H7, which has been interpreted (Heikkila et al., 1989) as further evidence indicating that transient down-regulation, rather than activation, of protein kinase C is indeed responsible for neurite outgrowth. In our hands, TPA induced a network of branching neurites resembling, but less pronounced than, that observed with staurosporine, and did not induce the longer neurites seen with staurosporine (not shown). Such neurite-promoting effects of phorbol esters, however, are restricted to treatments with relatively low concentrations; higher concentrations both inhibit outgrowth. and reverse H7-induced outgrowth (Felipo et al.. 1990; Minana et al.. 1990). Whether or not different protein kinase C inhibitors also induce the elaboration of morphologically different neurites in other cells, and any relationship of such a phenomenon to selective inhibition of different kinase C is oforms, remain to be determined. The data of the present study confirm and extend previous demonstrations of a regulatory role of protein kinase C in neuritogenesis. Although several agents which modulate this kinase’s activity can induce nel;ritogenesis. our results, and those of previous studies, demonstrate distinct morphological differences among the nature and extent of neurites obtained with each agent. Treatment of these cells for extended periods with retinoic acid (Shea. unpublished) induced the formation of neurites which resembled those elicited in the present study by H7, and lacked the branching network seen following staurosporine treatment. Neurites induced following treatment of cultured chick embryonic ganglion neurons with diacylglycerols, phospholipase C or muscarine chloride were morphologically distinct horn those induced by TPA (Hsu et al.. 1ox 0: Neurites elicited in mouse N 1XTG:! neuroblastoma and cultured
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cerebellar cells by treatment with H7 also differed morphologically from those obtained following dbcAMP-treatment (Tsuda et al., 1989). This situation is reminiscent of the different patterns of neurite formation elicited from mouse NB2a/dl cells treated with retinoic acid, dbcAMP, certain protease inhibitors or serum deprivation (Shea et al., 1985, 1988, 1990; Fischer et al., 1986). Recent studies have demonstrated that manipulation of the culture conditions, including alterations in culture substrate (Smalheiser and Schwartz. 1987; Smalheiser, 1989a,b), and the particular protease inhibitors and the sequence in which they are added (Shea et al., 1990) can selectively promote the elaboration of either transient, short neurites. or, conversely, stable, long neurites; moreover, particular treatments can, by mechanisms which remain undisclosed, convert transient processes into stable neurites (Smalheiser, 1989b). Nonwithstanding the complex series of events required for proper maturation and synaptogenesis, the axonal pathfmding, target innervation, initial, non-target mediated outgrowth of neurites also represents a critical phase of neuronal differentiation and, therefore. nervous system development. The elaboration of neurites as a result of protein kinase inhibition indicates that down-regulation of protein phosphorylation represents one key factor in this phenomenon. The advice
of Dr. Ubaldo Leli is gmtefrllly the Nntional
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acknowledged.
This r~esectn-h II’US .scq~porfed /I?
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Schubert D., Humphreys S., Baroni C. and Cohn M. (1969) In vitro differentiation of a mouse neuroblastoma.. Proc. Natl. Acad. Sci USA 64: 316-323. 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 USA 66:160-167. Shea, T.B., Fischer, 1. and Sapirstein, V.S. (1985) Effect of retinoic acid on growth and morphologic differentiation of NB2a neuroblas toma cells in culture Dev. Brain Res 2 I :307-3 14. Shea, T.B., Sihag, R.K. and Nixon, R.A. (1988) Neurofilament triplet proteins of NB2a/dI neuroblastoma: posttranslational modification and incorporation into the cytoskeleton during differentiation. Dev. Brain Res. 43:97-109. Shea, T.B., Beermann, M.L., and Nixon. R.A. (1990) Multiple proteases regulate neurite outgrowth. J. Neurochem. 21: in press. Shea, T.B. (1990) Neuritogenesis in mouse NB2a/dl neuroblastoma cells: trigerring by calcium inllux and involvement of actin and tubulin dynamics. Cell Biol. Int. Reports : in press Sidell, N., Altman, A., Haussler, M.R. and Seeger, R. (1983) Effects of retinoic acid on the growth and phenotypic expression of several human neuroblastoma cell lines. Exp. Cell Res. 148:21-30. Smalheiscr N.R. (1989a) Morphologic plasticity of rapid-onset neurites in NG108-IS cells stimulated by substratum-bound laminin. Dev. Brain Res. 45:39-47. Smalheiser N.R. (1989b) Analysis of slow-onset neurite formation in NGlOX-15 cells: implications for a unified model of neurite elongation. Dev. Brain Res. 45:49-57. Smalheiser N.R. and Schwartz N.B. (1987) Kinetic analysis of rapid-onset neuritc formation in NG108-15 cells reveals a dual role for substratum-bound laminin. Dev. Brain Res. 34: I1 I-121. Spinelli, W., Sonnenfield, K.H. and Ishii, D.N. (1982) Effects of phorbol ester tumorpromoters and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells. Cancer Res. 42: 5067-5073. Tamaoki. T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M. and Tomita. F. (1986) Staurosporine, a potent inhibitor of phospholipid/Ca ++-dependent protein kinase. Biochem. Biophys Res. Comm. 135:397-402. Tsuda, M., Ono. Katsuhiko.. Katayama, N., Yamagata, Y.. Kikuchi, K. and Tsuchiya, T. (lY89j Neurite outgrowth horn mouse neuroblastoma and cerebellar cells induced by the protcin kinasc inhibitor H7. Neurosci. Lett. 105:214-245. Tsuji, S.. Arita, M. and Nagai, Y. (1983) GQlb, abioactive ganglioside that exhibits novel nerve growth factor-kie activities in two neuroblastoma cell lines. J. Biochem. (Tokyo) 94:303306.
Paper
received
23.08.90.
Revised
paper
accepted
09.11.90.
International
Reports,
Vol. 75, No. 2, February
STAUROSPORINE-INDUCED DIFFERENTIATION OF HUMAN Thomas
MORPHOLOGICAL NEUROBLASTOMA
B. Shea1>2 and Mary
161
1991
CELLS
Lou Beermann
‘Ralph Lowell Laboratories, McLean Hospital, Belmont, MA 02178 2Depaxtment of Psychiatry. Harvard Medical School. Boston, MA 0211.5
ABSTRACT SH-SY-SY human neuroblastoma cells rapidly elaborated an extensive network ofneuritic processes following treatment with staurosporine, an inhibitor of protein kinase C. These neurites were retracted within 24hr following removal of inhibitor. Another inhibitor of protein kinase C, II7 ] l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], also induced rapid, reversible neurite outgrowth. However, neurites induced by these two inhibitors were morphologically distinct: staurosporine-treated cells elaborated a branching neuritic network adjacent to cell bodies, with some longer, unbranching neurites extending out of this network, while H7-treated cells elaborated only long, unbranching neurites. HA-1004 [N-(2guanidinoethyl)-5-isoquinolinesulfonamide], which inhibits of CAMP- and cGMP-dependent protein kinases but not protein kinasc C, did not induce neuritogenesis. Staurosporine-induced neurite outgrowth did not require protein synthesis but did require microtubule assembly. suggesting that cells contained the necessary components for neuritogenesis, and that alterations in protein phosphorylation alone was sufficient to initiate neurite outgrowth by rearrangement ot existing structures or cytoskeletal precursors. These results implicate phosphorylation in the regulation of neuronal differentiation and neuritogenesis.
INTRODUCTION Neuroblastoma cells undergo morphological and biochemical differentiation following treatment with a varitey of agents inducing compounds that raise CAMP levels (Prasad and Hsie, 1971; Shea et al., 1985, 1988), retinoic acid (Fischer et al., 1986; Pahlman et al., 1984; Shea et al., 1985; Sidell et al., 1983) serum deprival (Bottenstein and Sato, 1979; Seeds et al., 1970; Schubert et al., 1969, 1974; Shea et al.. 1985, 1990), certain protease inhibitors (Becherer and Wachsman, 1980; Cunningham and Gurwitz, 1989; Gibson et al., 1984; Gurwitz and Cunningham, 1988; Pittman et al., 1989; Saito and Kawashima, 1988; Sargent, 1989; Shea et al., 1990; Smalheiser, 1989a,b) increases in extracellular (Reboulleau, 1986) or intracellular (Anglister et al., 1982; Duprat and Kan, 1981; Gurevitch et al., 1982; Shea, 1990) calcium levels, gangliosides (Leskawa and Hogan, 1985; Roisen et al., 1981; Tsuji et al., 1983), and inhibitors of protein kinase C (Felipo et al., 1990; Hashimoto et al., 1989; Heikkila et al., 1989; Minana et al., 1990; Tsuda et al., 1989). By virtue of their ability to express many properties of differentiating and mature neurons following these treatments, neuroblastoma cells represent powerful model systems for studying the sequence of events underlying and accompanying the outgrowth and stabilization of neurites. The human neuroblastoma cell line, SH-SY-5Y has been reported to undergo morphological differentiation following treatment with the protein kinase C inhibitor H7 (Heikkila et al., 1989). Since H7 inhibits CAMP-dependent and cGMP-dependent kinases in addition to protein kinase C (Tsuji et al., 1983), however, we attempted to clarify the respective roles of these protein kinases in neuritogenesis. To accomplish this, we examined the effects on morphological differentiation of these cells following treatment with another protein kinase C inhibitor, staurosporine (Kase et al., 1987; Nakanishi et al., 1988; Ozaki et al.,
0309-l
651/91 I0201 61-6/$03.00/O
@ 1991 Academic
Press Ltd
162
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1987; Tamaokli et al., 1986), and HA1004, which inhibits CAMP-dependent and cGMP-dependent kinases but not protein kinase C (Hidaka et al., 1984). We observed that staurosporine and H7 induced morphologically distinct.neurites. but that HA1004 did not, confirming earlier suggestions that protein kinase C is involved in the regulation of neurite outgrowth. MATERIALS AND METHODS SH-SY-SY cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (FCS) at 370C in a humidified atmosphere containing 10% C02. Twenty-four hrs after plating, the cultures were treated with one or more of the following : staurosporine ( I-lOOpM), H7 (lo-500pM), HA-1004 (ZOOyM), colchicine ( IpM), and cycloheximide (OSpg/ml) for 6 and 24hrs. In some experiments, cultures treated for 6hrs were washed, the medium replaced with fresh medium lacking test compound(s), and incubation continued for a total of 24hrs. Stock solutions of inhibitors were prepared in 100% DMSO, then diluted in culture medium so that the tinal concentration of DMSO did not exceed 0.1%; this concentration did not induce morphological differentiation by 6hr, but did induce the outgrowth of short neurites by 24hrs of continuous treatment. Immediately after incubation, cultures were rinsed with phosphate-buffered saline (PBS: pH 7.4), fixed for ISmin at room temperature with 4% paraformaldehyde in O.lM phosphate buffer (pH 7.4), returned to PBS. then photographed and quantitated by phase-contrast microscopy (Shea et al., 1985). SH-SY-SY cells (originally obtained from the stocks of Dr. June L. Bielder, Memorial Sloan-Kettering Cancer Center, Rye, New York) were provided by Dr. Itzhak Fischer, E.K. Shriver Center, Waltham, MA, USA. Kinase inhibitors were generous gifts of Dr. Ubaldo Leli, McLean Hospital, Belmont MA, USA. All other reagents and chemicals were obtained from Sigma Chem. Co.(St. Louis, MO). RESULTS SH-SY-SY cells posses only short filopodia-like neurites; however, these cells rapidly elaborated an extensive network of neuritic processes following treatment with staurosporine (Fig. I). Despite this extensive outgrowth, these neurites were retracted within 24hr following removal of inhibitor. As previously reported, another inhibitor of protein kinase C, H7, also induced rapid neurite outgrowth (Heikkila et al., 1989; Hidaka et al., 1984). This outgrowth was also reversible following the removal of this compound. Although both inhibitors induced neurite outgrowth, staurosporine-treated cells elaborated a branching network of neurites, in addition to longer, unbranching neurites, while H7-treated cells elaborated mostly long,unbranching neurites, and lacked a similar network of shorter neurites. Quantitative analysis of this induction of differing neurite morphologies revealed that 85.4+15.1% of cells in staurosporine-treated cultures possessed a neuritic network along with one or more long, unbranching neurites, while only 11.3+7.9% of cells in H-7-treated cultures elaborated a network in addition to long unbranching neurites (p< 0.0005: Student’s t test).
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Figure 1: Induction of morphological differentiation of human neuroblastoma cells by inhibitors of protein kinase C. Phase-contrast micrographs of fixed SH-SY-SY cells cultured under the following conditions: Panel A, no additional additives for 24hrs ; Panel B, IpM s taurosporine for 6hrs. Arrows indicate the elaborate network of branching neurites near cell bodies; panel C, 1pM staurosporine for 24hrs; panel D, 1pM staurosporine for 6hrs followed by fresh medium without staurosporine for 20hrs. Note the loss of neurites; Panel E, 1pM H7 for 6hrs; panel F, 1pM H7 for 24hrs, arrows indicate a long, unbranching neurite. Note the lack of a neuritic network surrounding cell bodies; panel G, 1uM H7 for 4hrs followed by fresh medium without H7 for 20hrs. Note the loss of net&es; Panel H, 1OOpM HA-1004 for 6hrs. Neurites are not observed, panel C, 1OOpMHA-1004 for 24hrs. Note the presence of only short neurites; panel D, solvent (DMSO) alone for 24hrs. Note that short neurites are induced by treatment for 24hr with DMSO.
In contrast to the above results, HA-1004, which inhibits CAMP- and cGMP-dependent protein kinases but not protein kinase C (Hidaka et al., 1984), did not induce neuritogenesis over that observed for the solvent control (i.e., DMSO alone; see also Kimhi et al., 1976) when added at concentrations of up to 200 times those e&ctive for staurosporine and H7.
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Since treatment with protein kinase C inhibitors did not induce terminal differentiation of these cells, we examined whether or not these treatments induced alterations in genetic expression, or rather, the reorganization of existing materials. This was accomplished by protein syntheses inhibition by treatment with cycloheximide, and by prevention of microtubule polymerization by colchicine, during treatment with staurosporine (Fig. 2). Neurite outgrowth was unaffected by cycloheximide, indicating its independence of protein synthesis, but was blocked by colchicine, indicating the requirement for microtubule assembly as a driving force.
Firure 2: Neurites induced by protein kinase C inhibition are dependent upon microtubule assembly but independent of protein synthesis. Phase-contrast micrographs of fixed SH-SY-SY cells cultured under the following conditions: panel A, staurosporine for 6hrs; Panel B, cycloheximide for 90min followed by staurosporine and cycloheximide for 6 additional hrs; panel C, staurosporine and colchicine for 6hrs. Note that the staurosporine-induced elaboration 01 neurites is not inhibited by cyclohexime but is prevented by colchicine.
DISCUSSION Two inhibitors of protein kinase C, staurosporine and H7 induced the outgrowth of morphologically distinct neurites. Staurosporine-treated cells elaborated a branching network of neurites, in addition to longer, unbranching neurites, while H7-treated cells elaborated mostly long, unbranching neurites , and lacked a similar network of shorter neurites. These morphological differences may reflect the respective inhibition of different protein kinase C subtypes; although seven distinct subtypes of protein kinase C exist (Nishizuka, 1989). H7 selectively inhibits the alpha subtype (Pelosin et al., 1990), and the particular subtype(s) inhibited by staurosporine, unknown at present, may differ. The independence of staurosporine-mediated neurite outgrowth on protein synthesis, coupled with its requirement for microtubule assembly, indicates that
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the outgrowth of neurites induced by inhibition of protein kinase C is apparently mediated by the polymerization of cytoskeletal precursors or by a reorganization of existing cytoskeletal structures. A similar independence of the need for additional synthesis of RNA or protein has been reported for H7-mediated induction of neurites in N18TG2 neuroblastoma and cultured cerebellar cells (Tsuda et al., 1989). The data of the present study, and that of Tsuda and colleagues imply that specific phosphorylation events are sufficient to restrict the outgrowth of neurites. lnteres tingly. activator of protein kinase C, phorbol ester 12-Otetracedanoylphorbol-13-acetate (TPA) has previously been reported to induce limited outgrowth of neurites from SH-SY-SY cells (Akerman et al., 1984: Pahhnan et al., 1981; Spinelli et al., 1982: Heikkila et al., 1989) and other neuronal cell lines (Hsu et al., 1989). An explanation for the apparent discrepancy of how both inhibitors and activators of protein kinase C can induce neuritogenesis lies in the observation that, upon activation by TPA, protein kinase C associates with the plasma membrane, resulting in a transient downregulation of enzyme activity (Ballester and Rosen, 1985: Blackshear et al.. 1985: Krafi and Anderson, 1983). This line of reasoning is strengthened by the observation that mutant lines, including the human neuroblastoma IMR 33 (Jalava et al.. 1988). in which protein kinase C does not associate with the membrane following TPA-treatment do not exhibit morphological differentiation in the presence of TPA (Homma et al.. 1986). TPA-induced outgrowth ti-01x1 SK SY-SY cells was enhanced by the simultaneous addition of H7, which has been interpreted (Heikkila et al., 1989) as further evidence indicating that transient down-regulation, rather than activation, of protein kinase C is indeed responsible for neurite outgrowth. In our hands, TPA induced a network of branching neurites resembling, but less pronounced than, that observed with staurosporine, and did not induce the longer neurites seen with staurosporine (not shown). Such neurite-promoting effects of phorbol esters, however, are restricted to treatments with relatively low concentrations; higher concentrations both inhibit outgrowth. and reverse H7-induced outgrowth (Felipo et al.. 1990; Minana et al.. 1990). Whether or not different protein kinase C inhibitors also induce the elaboration of morphologically different neurites in other cells, and any relationship of such a phenomenon to selective inhibition of different kinase C is oforms, remain to be determined. The data of the present study confirm and extend previous demonstrations of a regulatory role of protein kinase C in neuritogenesis. Although several agents which modulate this kinase’s activity can induce nel;ritogenesis. our results, and those of previous studies, demonstrate distinct morphological differences among the nature and extent of neurites obtained with each agent. Treatment of these cells for extended periods with retinoic acid (Shea. unpublished) induced the formation of neurites which resembled those elicited in the present study by H7, and lacked the branching network seen following staurosporine treatment. Neurites induced following treatment of cultured chick embryonic ganglion neurons with diacylglycerols, phospholipase C or muscarine chloride were morphologically distinct horn those induced by TPA (Hsu et al.. 1ox 0: Neurites elicited in mouse N 1XTG:! neuroblastoma and cultured
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cerebellar cells by treatment with H7 also differed morphologically from those obtained following dbcAMP-treatment (Tsuda et al., 1989). This situation is reminiscent of the different patterns of neurite formation elicited from mouse NB2a/dl cells treated with retinoic acid, dbcAMP, certain protease inhibitors or serum deprivation (Shea et al., 1985, 1988, 1990; Fischer et al., 1986). Recent studies have demonstrated that manipulation of the culture conditions, including alterations in culture substrate (Smalheiser and Schwartz. 1987; Smalheiser, 1989a,b), and the particular protease inhibitors and the sequence in which they are added (Shea et al., 1990) can selectively promote the elaboration of either transient, short neurites. or, conversely, stable, long neurites; moreover, particular treatments can, by mechanisms which remain undisclosed, convert transient processes into stable neurites (Smalheiser, 1989b). Nonwithstanding the complex series of events required for proper maturation and synaptogenesis, the axonal pathfmding, target innervation, initial, non-target mediated outgrowth of neurites also represents a critical phase of neuronal differentiation and, therefore. nervous system development. The elaboration of neurites as a result of protein kinase inhibition indicates that down-regulation of protein phosphorylation represents one key factor in this phenomenon. The advice
of Dr. Ubaldo Leli is gmtefrllly the Nntional
Scierm
acknowledged.
This r~esectn-h II’US .scq~porfed /I?
Fouruiatlorl (BNS 8910869/.
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Paper
received
23.08.90.
Revised
paper
accepted
09.11.90.