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© The Rockefeller University Press, 0021-9525/1998//1625 $5.00
The Journal of Cell Biology, Volume 141, Number 7, , 1998 1625-1636

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

Masaya Hirose^*, Toshimasa Ishizaki^*, Naoki Watanabe^*, Masayoshi Uehata, Onno Kranenburg, Wouter H. Moolenaar, Fumio Matsumura^||, Midori Maekawa^*, Haruhiko Bito^*, and Shuh Narumiya^*

^* Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8315, Japan; Discovery Research, Yoshitomi Pharmaceutical Industries, Iruma, Saitama 358-0026, Japan; Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands; and ^|| Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1059

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.

Abbreviations used in this paper: GFAP, glial fibrillary acidic protein; LPA, lysophosphatidic acid; MLC, myosin light chain; p160ROCK, p160 Rho-associated coiled coil-forming protein kinase.

Address all correspondence to Shuh Narumiya, Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8315, Japan. Tel.: (81) 75-753-4392. Fax: (81) 75-753-4693. E-mail: snaru{at}mfour.med.kyoto-u.ac.jp

AXONAL guidance is regulated by interaction of a growth cone with various environmental cues. Extracellular signals critical in this process include diffusible chemoattractants and chemorepellants, or various kinds of extracellular matrix proteins and cell adhesion molecules. Once the growth cone receives these signals, it moves either toward or away from them (for review see Keynes and Cook, 1995). Such signal-induced motion is, at least in part, mediated by protrusion and retraction of the filopodia and lamellipodia present in the growth cone. The shape and movement of these structures is presumably determined by the reorganization of the actin cytoskeleton (for review see Bently and O'Connor, 1994; Lin et al., 1994; Tanaka and Sabry, 1995).

Rho family proteins including Rho, Rac, and Cdc42 participate in the reorganization of actin cytoskeleton from yeast to mammals (for review see Hall, 1994). In cultured fibroblasts, Rho regulates the formation of focal adhesions and stress fibers, whereas Rac and Cdc42 regulate the growth factor–induced membrane ruffling and filopodia formation, respectively (Ridley and Hall, 1992; Ridley et al., 1992; Kozma et al., 1995; Nobes and Hall, 1995). Recently, several lines of evidence suggested that Rho family proteins play a critical role in axonal and dendritic outgrowth. In Drosophila melanogaster, constitutively active and dominant-negative Rac mutants both induced the disruption of axon outgrowth (Luo et al., 1994). Furthermore, axon terminal formation was significantly reduced in Purkinje cells of mice expressing a constitutively active form of Rac as a transgene (Luo et al., 1996). mig-2 gene product, also a member of the Rho family proteins, was shown to be critical for cell migration and axon outgrowth in Caenorhabditis elegans (Zipkin et al., 1997). In vitro, microinjection of Cdc42 and Rac into cultured N1E-115 neuroblastoma cells, respectively, induced filopodia and lamellipodia in the growth cone (Kozma et al., 1997); these GTPases mediated the action of acetylcholine to induce these membrane structures in this cell line. On the other hand, other types of agonists such as lysophosphatidic acid (LPA),¹ thrombin, sphingosine-1-phosphate, or serum itself induced rapid growth cone collapse and neurite retraction in several cultured neuronal cell lines such as N1E-115 cells, NG108-15 neuroblastoma-glioma hybrid cells, and PC12 pheochromocytoma cells (Jalink et al., 1992, 1994; Postma et al., 1996; Tigyi et al., 1996a). This dramatic morphological change was inhibited by pretreatment of the cells with botulinum C3 exoenzyme, which inactivates Rho by ADP ribosylation, indicating the involvement of Rho in this process. Consistently, overexpression or microinjection of constitutively active Rho into these cells induced growth cone collapse and marked cell rounding (Kozma et al., 1997; Kranenburg et al., 1997). These findings supported the hypothesis that the Rho family GTPases mediated the protrusion and the collapse of the growth cone and perhaps regulated its motility. However, the intracellular signaling mechanisms by which these GTPases exert their actions in the growth cone have remained unknown.

Recently, several putative target molecules of Rho were isolated (for review see Narumiya et al., 1997). Among them, a family of Rho-associated serine/threonine kinase isozymes named p160ROCK (Ishizaki et al., 1996) and ROK/Rho-kinase/ROCK-II (Leung et al., 1995; Matsui et al., 1996; Nakagawa et al., 1996) has been identified as a new class of Rho effectors, which induced focal adhesions and stress fibers in cultured fibroblasts and epithelial cells (Leung et al., 1996; Ishizaki et al., 1997; Amano et al., 1997). Kimura et al. (1996) showed that Rho-kinase phosphorylated and inactivated myosin phosphatase in vitro and suggested that it thereby regulated myosin light chain (MLC) phosphorylation. We recently identified a new pyridine derivative named Y-27632 as a specific inhibitor of the ROCK/ROK family of protein kinases (Uehata et al., 1997). This compound inhibited smooth muscle contraction both in vitro and in vivo, as well as the formation of stress fibers and focal adhesions induced by p160ROCK in cultured cells. These findings suggested that ROCK mediated the Rho effect on the formation of focal adhesions and stress fibers, at least partly through the regulation of actomyosin system.

In this study, we used Y-27632 and wild-type and a dominant-negative form of p160ROCK, and examined the role of p160ROCK in the neurite remodeling of N1E-115 cells. Our findings indicate that p160ROCK not only mediates the agonist-induced neurite retraction but also tonically suppresses neurite outgrowth in the presence of serum. The former action appears to be exerted through both the activation of the actomyosin system and the disassembly of the intermediate filaments and microtubules. The latter suppression appears partly due to inactivation of Cdc42 and Rac, because the inhibition of the p160ROCK leads to the activation of Cdc42 and Rac and to the neurite induction. Our findings further indicate that the opposing actions of p160ROCK on the actin cytoskeleton and the other two types of cytoskeletons are not limited to the cells of neuronal origin.

	Materials and Methods

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Materials
LPA was purchased from Sigma Chemical Co (St. Louis, MO). Rabbit polyclonal anti-peripherin antibody, mouse monoclonal anti-vimentin antibody (LN-6), and mouse monoclonal anti–β-tubulin antibody (N-357) were purchased from Chemicon (Temecula, California), Sigma Chemical Co., and Amersham (Little Chalfont, UK), respectively. Mouse monoclonal anti-FLAG M2 antibody was obtained from Eastman Kodak Co. (New Haven, CT). pEXV-myc-V14RhoA and Swiss 3T3 cells were kindly provided by A. Hall (University College London, London, UK). pCMV-myc-N17Cdc42 and pCMV5-FLAG-N17Rac1 were provided by M. Symons (Onyx Pharmaceuticals, Richmond, CA) and Y. Kaziro (Tokyo Institute of Technology, Tokyo, Japan). Y-27632 was supplied by Yoshitomi Pharmaceutical Industries (Saitama, Japan). Botulinum C3 exoenzyme was purified as described (Jalink et al., 1994). Rabbit polyclonal anti-ROCK antibody 20490 and anti-myc antibody 9E10 were prepared as described (Fujita et al., 1997; Ishizaki et al., 1997).

Assay for Neurite Retraction
Mouse neuroblastoma N1E-115 cells were maintained in DME supplemented with 10% FBS. For neurite induction, N1E-115 cells were cultured in serum-free DME for 24 h. More than 90% of the cells became flattened and extended neurites under these conditions. Neurite retraction was evoked by incubating cells with 1 µM LPA for 10 min, with or without preincubation with indicated concentrations of Y-27632 for 30 min. Phase-contrast photomicrographs were taken before the Y-27632 pretreatment and after the LPA stimulation. More than 200 cells were identified in randomly chosen fields of view, and the percentage of cells that retracted neurites after LPA application was calculated.

p160ROCK Expression Constructs and Cell Transfection
Myc-tagged wild-type and dominant–negative mutant of p160ROCK (KD-IA) were constructed in the pCAG mammalian expression vector as previously described (Ishizaki et al., 1997) (see Fig. 1). N1E-115 cells were plated at a density of 2 x 10⁴ cells per 3.5-cm dish and of 10⁵ cells per 3.5-cm dish for immunofluorescence and immunoblotting studies, respectively. After culture for 1 d, cells were transfected with the plasmid DNA (1 µg) by the application of lipofectamine-DNA coprecipitates in Opti-MEM (Gibco Laboratories, Grand Island, NY) (Ishizaki et al., 1996). For cotransfection of KD-IA with either N17Rac1 or N17Cdc42, 1 µg of each plasmid DNA was used. For transfection with V14Rho, wild-type p160ROCK, the medium was changed to serum-free DME at 4 h and then the cells were cultured for another 12 h. For transfection with the KD-IA mutant, the medium was replaced by DME containing 10% FBS at 4 h and the cells were cultured for another 16 h in the presence of serum.

View larger version (8K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Schematic representation of the wild-type and the KD-IA mutant of p160ROCK. Domains of p160ROCK and the point mutations in the KD-IA mutant are illustrated. KD, serine/threonine protein kinase domain; CCD, coiled-coil forming amphiphatic -helical domain; RBD, Rho-binding domain; PH, pleckstrin homology domain; CRD, cysteine-rich zinc-finger domain; X, positions of mutations with amino acid numbers.

Microinjection and Video Microscopy
Swiss 3T3 cells were plated on a glass coverslip at 3 x 10⁴ cells per 3.5-cm dish and then cultured in DME containing 10% FBS for 24 h. Medium was changed to Hepes-buffered DME containing 10% FBS and the cells were incubated for 1 h. A coverslip was then transferred onto a heated stage maintained at 37°C in the Hepes-buffered medium. C3 exoenzyme at 150 µg/µl was injected into Swiss cells by the use of Eppendorf Microinjector 5242 (Eppendorf Scientific, Inc., Hamburg, Germany) as described previously (Watanabe et al., 1997). Time-lapse analysis was carried out by phase contrast on an Axiovert microscope (model 100; Carl Zeiss, Inc., Thornwood, NY) with a Hamamatsu charge-coupled device camera (model C2400; Hamamatsu Phototonics, Hamamatsu City, Japan) and recorded by a SONY laser video disc recorder (model LVR-3000AN; Sony Corp., Park Ridge, NJ) Images were printed by a SONY color video printer (model UP-1850).

Western Blotting Analysis of p160ROCK in N1E-115 Cells
N1E-115 cells were lysed in Laemmli's SDS-PAGE sample buffer. The cell lysates were subjected to SDS-PAGE on a 8% polyacrylamide gel, and then separated proteins were transferred onto a nitrocellulose membrane (BA85; Schleicher & Schuell, Inc., Keene, NH). The membrane was blocked with 5% BSA in Tris-buffered saline. The membrane was probed with anti-p160ROCK rabbit antibody 20490 (Fujita et al., 1997) and then the signal was detected by alkaline phosphatase-conjugated anti–rabbit IgG antibody (Kirkegaard & Perry Laboratories, Inc.).

Detection of MLC Phosphorylation
N1E-115 cells were allowed to extend neurites in serum-free DME and were preincubated with or without various concentrations of Y-27632 for 30 min. The cells were then stimulated for indicated times with 1 µM LPA and then lysed in Laemmli's SDS-PAGE sample buffer. The cell lysates were subjected to immunoblotting with anti-phosphorylated MLC (p-MLC) rabbit antibody (Matsumura et al., 1998) using alkaline phosphatase staining system as described above.

	Results

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Y-27632 Inhibits Agonist-induced Neurite Retraction
Under serum starvation, most N1E-115 cells became flattened and extended neurites with prominent growth cones (Fig. 2, B and D). As reported previously (Suidan et al., 1992; Jalink et al., 1993), the addition of LPA to these cells caused rapid collapse of growth cones and neurite retraction. This morphological change was usually complete within 5 min, and in 10 min most cells became round and few, if any, neurites remained extended (Fig. 2 C). We previously demonstrated that this neurite retraction was blocked completely by a previous treatment of the cells with C3 exoenzyme, indicating the involvement of Rho in this process (Jalink et al., 1994).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Western blotting of p160ROCK in N1E-115 cells (A) and inhibition of LPA-induced neurite retraction and cell rounding by Y-27632 (B–E). (A) N1E-115 cells were lysed in Laemmli– SDS-PAGE sample buffer and then subjected to immunoblotting using anti-p160ROCK antibody. (B–E), N1E-115 cells were maintained in serum-free DME for 24 h and allowed to extend neurites. The cells were pretreated without (B and C) or with 10 µM Y-27632 (D and E) for 30 min, and exposed to 1 µM LPA for 10 min. The same fields were photographed before the drug addition (Band D) and after LPA stimulation (C and E). Bar, 100 µm.

View larger version (7K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Concentration-dependent inhibition by Y-27632 of LPA-induced neurite retraction in N1E-115 cells. Serum-starved N1E-115 cells were pretreated with the indicated concentrations of Y-27632 for 30 min and then exposed to 1 µM LPA for 10 min. The percentages of cells with complete neurite retraction are shown. Results of three experiments are shown as mean ± SEM.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Morphology of N1E-115 cells expressing active Rho and p160ROCK. N1E-115 cells were transfected with V14Rho (A and B) or wild-type p160ROCK (C and D), and were cultured in serum-free DME for 12 h. Overexpressing cells were identified by the positive Myc-staining (A and C) and their F-actin structures were examined (B and D). Note that the transfected cells do not extend neurites even under serum-starved conditions. In addition, marked bleb formation was also noted in some of p160ROCK-expressing cells. Bar, 20 µm.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Morphology of N1E-115 cells expressing the KD-IA mutant and of N1E-115 cells treated with Y-27632. (A) N1E-115 cells were transfected with the KD-IA mutant, a dominant-negative form of p160ROCK, and were cultured for 16 h in the presence of 10% FBS. The cells were double-stained for the Myc-tag (a) and F-actin (b). Note that the transfected cells extend long neurites in the presence of serum. (B) N1E-115 cells were cultured in the presence of 10% FBS, and treated with 10 µM Y-27632, a specific ROCK inhibitor. Phase-contrast photomicrographs were taken at 0 (a) and 60 min (b) after the addition of the compound. Bar, 20 µm.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Inhibition by N17Cdc42 and N17Rac of the neurite generation induced by the KD-IA mutant of p160ROCK. N1E-115 cells were transfected with KD-IA p160ROCK alone (A and B), with N17Cdc42 and KD-IA p160ROCK (C and D) or with N17Rac and KD-IA p160ROCK (E and F). The transfected cells were cultured in DME containing 10% FBS for 16 h. Overexpressing cells were identified with p160ROCK staining (A, C, and E) and F-actin was stained with phalloidin (B, D, and F). Bar, 20 µm. (G) Quantification of cells bearing no neurites, short neurites, and long neurites. N1E cells expressing KD-IA alone, KD-IA and N17Cdc42, or KD-IA and N17Rac were identified as above, and the numbers of cells bearing no neurites, or neurites shorter or longer than 100 µm were determined. *, P < 0.001 compared with KD-IA.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 7 LPA-induced MLC phosphorylation and its inhibition by Y-27632 in N1E-115 cells. (A) Time course of LPA-induced MLC phosphorylation. Serum straved N1E-115 cells were incubated with 1 µM LPA for the indicated times. Cells were collected in Laemmli-SDS-PAGE sample buffer and then subjected to immunoblotting using anti-phosphoMLC antibody. (B) Concentration-dependent inhibition of MLC phosphorylation by Y-27632. Serum-starved N1E-115 cells were treated with the indicated concentrations of Y-27632 for 30 min, and then stimulated with LPA for 2 min. The cells were lysed and analyzed as above. The content of MLC in the cells did not change during LPA stimulation or Y-27632 treatment.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Peripherin and tubulin staining of serum-starved, serum-fed, and KD-IA–transfected N1E-115 cells. N1E-115 cells were cultured in the absence (A and D) or presence (B and E) of serum, or were transfected with the KD-IA mutant of p160ROCK and cultured in DME containing 10% serum (C and F). The cells were stained with either anti-peripherin (A, B, and C) or anti-tubulin (D, E, and F) antibodies. In C and F, the cells were also stained with either anti-myc antibody (C), or anti-ROCK antibody (F), and the cells expressing KD-IA protein were identified (indicated by arrows). Images built from optical sections by a confocal imaging system are shown. Bar, 20 µm.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Vimentin staining of Swiss 3T3 cells. Swiss 3T3 cells were cultured in the presence (A and B) or absence (C and D) of serum, or with 30 µg/ml C3 exoenzyme for 72 h (E and F) or 10 µM Y-27632 for 2 h (G and H) in the presence of serum, and were stained with OregonGreen phalloidin (A, C, E, and G) or with anti-vimentin antibody (B, D, F, and H). Images built up from optical sections by a confocal imaging system are shown. Bar, 20 µm.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 11 Tubulin staining of Swiss 3T3 cells. Swiss 3T3 cells were cultured in the presence (A and B) or absence (C and D) of serum, or with C3 exoenzyme (E and F) or Y-27632 (G and H) in the presence of serum, and were stained with OregonGreen phalloidin (A, C, E, and G) or with anti-tubulin antibody (B, D, F, and H). Images built from optical sections by a confocal imaging system are shown. Bar, 20 µm.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Video-microscopy of Swiss 3T3 fibroblast microinjected with C3 exoenzyme. Swiss cells were maintained on a glass coverslip in Hepes-buffered DME containing 10% FBS and microinjected with C3 exoenzyme. Morphology of the injected cells was monitored by time-lapse video-microscopy. Four images taken every two min are shown. Time after the injection is indicated in the top left corner of each image. Note cell processes change in their morphology in a time-dependent manner; arrowhead, time-dependent elongation of one process. Bar, 20 µm.

	Discussion

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Our previous study (Jalink et al., 1994) indicated the generation of tension along the neurites in N1E-115 cells exposed to LPA. In this study, we have shown that LPA application increased the level of phosphorylated MLC, with a time course overlapping with that of the LPA-induced morphological changes. Furthermore, Y-27632 inhibited both the phosphorylation and morphological changes with a similar dose-response relationship. Since MLC phosphorylation is a critical step in generating an actomyosin-based contractile force, our results suggest an essential role for p160ROCK in triggering a Rho-dependent contraction in N1E-115 cells, thus presumably leading to neurite retraction.

The contraction of the actomyosin system in smooth muscle and nonmuscle cells is thought to be regulated by two mechanisms: (a) one is dependent on increase in free Ca²⁺ ion in the cell and is mediated by MLC kinase and (b) the other is a Ca²⁺ sensitization mechanism. Several lines of evidence now indicate a role for the ROCK/ROK kinase family in the latter mechanism. Kimura et al. (1996) reported that Rho-kinase phosphorylates the myosin binding subunit of myosin phosphatase in vitro and thereby inhibits its activity. Amano et al. (1996) further suggested the possibility that Rho-kinase may also directly phosphorylate MLC. Finally, Uehata et al. (1997) demonstrated that Y-27632, which blocks the ROCK/ROK kinases in vitro, inhibited smooth muscle contraction in vivo by specifically acting on the Ca²⁺-sensitization mechanism. Our present work extends these findings and provides a common basis for establishing an essential role for p160ROCK in catalyzing the Rho-dependent regulation of actomysion-based contractility, in neuronal as well as in nonneuronal cell types.

The Rho–ROCK Pathway Regulates Reorganization of the Microtubules and the Intermediate Filaments
It is well known that the intermediate filaments and the microtubules play important roles in the formation of neurites or similar processes. Microtubule depolymerizating drugs can cause neurite collapse in many types of cultured neuronal cells (Daniels, 1975; Solomon and Magendantz, 1981; Joshi et al., 1985). In PC-12 cells, exposure to tubulin mRNA antisense oligonucleotides led to tubulin depletion and inhibition of neurite outgrowth (Teichman-Weinberg et al., 1988). As for the intermediate filaments, a similar antisense mRNA strategy was used to demonstrate the requirement for glial fibrillary acidic protein (GFAP) in the formation of stable processes in human astrocytoma cells (Weinstein et al., 1991).

The present study has demonstrated that in N1E-115 cells, inhibition of ROCK in the presence of serum, by overexpressing a dominant-negative form of p160ROCK, generated an outgrowth of neuritic processes, which were filled with thick fibers immunopositive for tubulin and peripherin (refer to Fig. 8). We also showed that in Swiss 3T3 cells, treatments with C3 exoenzyme or Y-27632, by themselves, induced extension of long neurite-like processes, positively stained for structures containing both tubulin and vimentin, a predominant intermediate filament in this cell type. These results suggest that the ROCK/ROK protein kinase family may be critically involved in the regulated reorganization of the intermediate filaments and the microtubules, not only in neuronal but also in nonneuronal cells. Our findings are consistent with Paterson et al. (1990) who showed that the microinjection of V14Rho caused the collapse of the vimentin structure in Swiss 3T3 cells. Thus, the morphological phenotypes and signal transduction pathways regulated by p160ROCK may be quite similar in neuronal cells such as N1E-115 cells and nonneuronal cells such as Swiss 3T3 cells. However, the ultimate morphology of two types of cells appears different; marked cell rounding in neuronal cells and flattened cell bodies with stress fibers in nonneuronal cells. This is likely due to the difference in the integrin-based focal adhesions of these cells. How p160ROCK is involved in the differential regulation of these structures in the two types of cells remains to be investigated.

At present, we do not know whether the ROCK effect on intermediate filaments is a direct or indirect consequence of phosphorylation events mediated by ROCK/ ROK kinases. Phosphorylation of the intermediate filaments has been shown to be required for their interaction with other cytoskeletal components and has been implicated in neurite extension and functions (Nixon et al., 1987; Aletta et al., 1989). Kosako et al. (1997) reported that Rho-kinase, an isozyme of p160ROCK, phosphorylated a specific residue in GFAP, that had been previously associated with the disassembly of the GFAP filaments during cytokinesis in astrocytes. Thus, it is possible that activated p160ROCK directly phosphorylates different types of intermediate filament proteins, even in the cell types that we examined, namely N1E-115 and Swiss 3T3 cells. Our preliminary analysis, however, did not detect enhanced phosphorylation of peripherin after LPA stimulation of N1E-115 cells, although we could not exclude the possibility that residual p160ROCK activity could phosphorylate a specific residue of this protein (Hirose, M., T. Ishizaki, and S. Narumiya, unpublished observations). On the other hand, the disassembly of intermediate filaments as well as microtubules may well be a secondary consequence of the regulation of the actomyosin system, in which the tension generated by the ROCK-induced increase in the myosin-based contractility may generate a yet unknown secondary negative signaling event, which then acts on the cytoskeletons and leads to their disruption. As discussed below, many lines of evidence suggest that a positive regulation of the neurite outgrowth triggered by inactivation of Rho may be exerted by other members of the Rho family GTPases, such as Cdc42 and Rac. Thus, Rho-dependent regulation of the assembly and disassembly of the intermediate filaments might in fact be controlled via a reciprocal activation/inactivation processes between Rho and Cdc42/Rac.

The present study has also shown that activation and inactivation of p160ROCK regulates the disassembly and assembly of the microtubules. Previous reports demonstrated that microtubule-depolymerizing agents induced stress fiber formation in a C3 exoenzyme-sensitive manner, thereby suggesting a signaling pathway from disruption of microtubules to Rho (Danowski et al., 1989; Enomoto, 1996; Zhang et al. 1997). However, no pathway from Rho to the microtubules has been indicated. Our study represents the first formal demonstration of a significant role for a Rho-associated molecule, p160ROCK, in this process. The parallelism in the p160ROCK-dependence seen in both intermediate filaments and microtubules indicate that Rho may simultaneously regulate the assembly and disassembly of both cytoskeletal components through a similar mechanism. In any event, the present study raises an interesting possibility that the regulatory role of Rho GTPases on the cytoskeleton may extend to the reorganization of the intermediate filaments and microtubules, in addition to the well-known roles in actin cytoskeleton. Current efforts in our laboratories are directed towards the identification of the ROCK substrates implicated during neurite retraction process, in order to sort out the distinct signaling pathways involved in these multiple regulatory processes.

Signaling Pathways in Neurite Remodeling: Interaction of the Rho–ROCK Pathway with Other Pathways
The present study has demonstrated a critical role for the Rho-ROCK pathway not only in the agonist-induced neurite retraction, but also in the tonic inhibition of neurite outgrowth in N1E-115 cells in the presence of serum. Suppression of this inhibitory pathway releases the cells from their constraint and enables them to sprout and extend new neurites. This role of the Rho–ROCK pathway as a tonic negative regulator in cell process formation appears not to be limited to N1E-115 cells, because previous studies demonstrated that inactivation of Rho by botulinum C3 exoenzyme could also induce long neurites in PC12 cells (Nishiki et al., 1990; Tigyi et al., 1996a) as well as in neuroblastoma NG108-15 cells (Moolenaar, 1994). Kozma et al. (1997) found that this apparently stimulatory effect of C3 exoenzyme on neurite outgrowth in N1E-115 cells could be inhibited by expressing dominant-negative forms of Cdc42 and Rac. Conversely, activation of Cdc42 and Rac stimulated neurite outgrowth by inducing filopodia and lamellipodia in the growth cones. Furthermore, coinjection of Rho with Cdc42 blocked the Cdc42-induced formation of filopodia, suggesting that Rho signaling downregulates Cdc42 signaling in N1E-115 cells in a dominant fashion (Kozma et al., 1997). Similarly, the overexpression of the guanine nucleotide exchange factor for Rac, Tiam1, induced neurite-like processes, which could be antagonized by expression of constitutively active Rho (van Leeuwen et al., 1997). Taken together, these findings indicate that p160ROCK, as a downstream effector of Rho, may negatively regulate the Cdc42 and Rac pathway, both of which play a positive role in growth cone expansion. Consistent with this idea, we found that expression of N17Cdc42 and N17Rac, dominant-negative forms of Cdc42 and Rac respectively, prevented the neurite growth induced by the dominant-negative form of p160ROCK (refer to Fig. 6).

In addition to these signaling cross-talks at the GTPases level, functional interaction between different kinase pathways may also have to be considered. It has been known for some time that activation of the cAMP pathway induces neurite outgrowth in various neuronal cells including PC12 cells (for example see Gunning et al., 1981). Recently, Tigyi et al. (1996b) has reported that LPA-induced neurite retraction is inhibited by the elevation of cAMP levels in PC12 cells. This effect was not seen in a mutant PC-12 clone deficient in protein kinase A. These results suggest that the Rho–ROCK pathway may be antagonized by the cAMP signaling pathway during ligand-induced neurite remodelling and that inhibition of neurite retraction by cAMP may be exerted by an A kinase–dependent inhibition of the Rho–ROCK pathway. Whether the cyclic AMP-PKA effect acts on Rho, ROCK, a ROCK substrate, or further downstream remains to be determined. The possible interaction of these signaling pathways is depicted in Fig 12.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 12 Model of signal transduction and cross-talks of the Rho–ROCK pathway in the neurite remodeling. p160ROCK is activated downstream of Rho in response to agonist stimulation, and in turn induces the actomyosin-based contractility and the disassembly of the microtubules and the intermediate filaments, leading to the neurite retraction. p160ROCK also transmits a negative signal to the Cdc42/Rac pathways and suppresses the neurite outgrowth by tonically inhibiting their actions. The activation of the cAMP–A kinase pathway is likely to inhibit the Rho–ROCK pathway and to release the suppression of neurite outgrowth. Disassembly of intermediate filaments may be caused by direct phosphorylation by p160ROCK at specific amino acid residue(s) of this cytoskeletal proteins as shown for GFAP (Kosako et al., 1997) or may well be a secondary consequence of ROCK's effect on the actomyosin system.

An independent report also suggested an involvement of Rho-kinase–mediated MLC phosphorylation in LPA-induced neurite retraction in N1E-115 cells. (Amano, M., K. Chihara, N. Nakamura, Y. Fukata, T. Yano, M. Shibata, M. Ikebe, and K. Kaibuchi. 1998. Genes Cells 3:177–188).

	Acknowledgments

 
The authors thank K. Itoh and K. Yoshioka (Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan) for advice on MLC phosphorylation, P. Madaule (Kyoto University, Kyoto, Japan) for discussion, and K. Nonomura and T. Arai for technical and secretarial assistance, respectively. M. Hirose is on leave from the Department of Obstetrics and Gynecology, Shiga University of Medical Science, and thanks its chairman Y. Noda for encouragement.

This work was supported in part by a Grant-in-aid for Specially Promoted Research (08102007) from the Ministry of Education, Science, and Culture of Japan, and grants from Human Frontier Science Program, from the Japanese Foundation on Metabolism and Diseases, and from the Kyoto University Academic Research Award.

Submitted: 25 February 1998

Revised: 14 May 1998

M. Hirose and T. Ishizaki contributed equally to this work.

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