Article |
Address correspondence to Toshihide Yamashita, Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3221. Fax: 81-6-6879-3229. E-mail: tyama{at}anat2.med.osaka-u.ac.jp
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Abstract |
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Key Words: p21Cip1/WAF1; Rho-kinase; neurite outgrowth; differentiation; Rho
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Introduction |
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During the course of differentiation of the neuronal cells, p21Cip1/WAF1 also plays important roles in regulating the cell cycle. In several cell lines during differentiation after nerve growth factor treatment, the expression of p21Cip1/WAF1 protein was increased (Decker, 1995; Dobashi et al., 1995; Yan and Ziff, 1995; Poluha et al., 1996; van Grunsven et al., 1996; Gollapudi and Neet, 1997; Erhardt and Pittman, 1998). However, neurons after differentiation seem to have special features, distinct from other cell types, as newborn neurons extend axons and dendrites to communicate with appropriate targets. For example, dorsal root ganglion neurons up to postnatal day 3 to 4 or embryonic retinal ganglion neurons can extend their neurites rapidly on myelin-associated glycoprotein, which is an effective neurite outgrowth inhibitor for adult neurons (Johnson et al., 1989; Mukhopadhyay et al., 1994; De Bellard et al., 1996; Cai et al., 2001). These findings suggest that immature neurons may have intrinsic mechanisms that confer resistance to the inhibitory molecules.
Here we show a novel function of cytoplasmic p21Cip1/WAF1. Cytoplasmic expression of p21Cip1/WAF1 was observed in newborn neurons that extensively extend neurites. As p21Cip1/WAF1 binds to Rho-kinase and inhibits its activity, changes in the cytoskeletal organization are at least partly attributable to the nonenzymatic protein inhibitor.
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Results |
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In vitro differentiation of N1E-115 cells is associated with p21Cip1/WAF1 expression in the cytoplasm
We next used neuroblastoma N1E-115 cells to examine whether neuronal differentiation was associated with cytoplasmic expression of p21Cip1/WAF1. N1E-115 cells, which were induced to differentiate by DMSO, were immunostained with the anti-p21Cip1/WAF1 antibody. After 24 h of DMSO treatment, p21Cip1/WAF1 was induced in the nucleus (Fig. 2 B). However, after 4 d, a time point when the extensive neurite genesis was well evident, p21Cip1/WAF1 was mainly localized in the cytoplasm (Fig. 2 C). In this regard, the differentiation-associated cytoplasmic expression of p21Cip1/WAF1 is not restricted to chick retinal precursor cells.
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The lack of an interaction of the full-length-p21 with Rho-kinase may be attributable to the difference of the localization in the cells. Therefore, we tested the in vitro interaction of the recombinant full-length p21Cip1/WAF1 and Rho-kinase. These proteins in vitro bound to each other (Fig. 5 D). As GST fused to the fragment of Rho kinase used here corresponds to the catalytic region of Rho-kinase (GSTCAT; aa 6553), p21Cip1/WAF1 may directly bind to the catalytic region of Rho-kinase. This is substantiated by our finding that S6 kinase substrate peptide (AKRRRLSSLRA) as well as Y-27632 inhibited the interaction of p21Cip1/WAF1 with Rho-kinase dose dependently (Fig. 5 D). These results suggest that p21Cip1/WAF1 associates with Rho-kinase in the cytoplasm.
p21Cip1/WAF1 inhibits Rho-kinase activity
We next investigated whether p21Cip1/WAF1 could inhibit the activity of Rho-kinase in vitro. The kinase assay was carried out using S6 kinase substrate peptide and [-32P] ATP. By using a scintillation counter, the quantity of 32P-labeled substrate peptide on the phosphocellulose paper was determined. This kinetic analysis revealed that p21Cip1/WAF1 inhibited the Rho-kinase activity toward S6 kinase substrate peptide in a dose-dependent manner (Fig. 6 A), and the IC50 value estimated was 1.43 nM.
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Cytoplasmic p21Cip1/WAF1 promotes neurite outgrowth and branching of the hippocampal neurons
To investigate the relevance of our findings that the cytoplasmic p21Cip1/WAF1 acts on Rho-kinase, we assessed the effects on neurons. Cultures of the hippocampal neurons from rat E18 embryos were used. We chose these neurons, as they did not express endogenous p21Cip1/WAF1 enough to be detected by immunocytochemistry using the anti-p21Cip1/WAF1 antibody (unpublished data). Dissociated hippocampal neurons were incubated for 48 h and transfected with NLS-p21. 24 h after transfection, the cells were fixed and immunolabeled with ß-tubulin III. The total neurite length per neuron, the axonal length, defined as the length of the longest neurite per neuron, the number of primary processes originating from the neuronal somata, and the number of branch points per neuron were determined (Neumann et al., 2002). The neuronal morphology of the cells expressing
NLS-p21 was apparently different from the control cells without transfection or expressing GFP (Fig. 7 A). The cells with the
NLS-p21 expression extended longer neurites and had more branch points than the control cells (GFP-expressing cells or no transfection). Ectopic expression of
NLS-p21 increased the total neurite length per neuron from 135.9 µm (±7.2 µm SEM) to 307.2 µm (±34.0 µm SEM), the axonal length from 66.3 µm (±3.2 µm SEM) to 162.9 µm (±18.6 µm SEM), and the number of branch points per neuron from 1.3 (±0.2 SEM) to 2.6 (±0.3 SEM). However, no change in the number of primary processes was found by overexpression of cytoplasmic p21Cip1/WAF1 (Fig. 7 B). These results indicate that cytoplasmic p21Cip1/WAF1 regulates neurite remodeling in the embryonic hippocampal neurons.
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Discussion |
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Cytoplasmic expression of p21Cip1/WAF1
There have been several reports that show cytoplasmic expression of p21Cip1/WAF1 and its possible mechanisms. In peripheral blood monocytes and differentiating U937 cells, p21Cip1/WAF1 was detected in the cytoplasm (Lubbert et al., 1991; Asada et al., 1999). U937 cells can differentiate into monocytes with vitamin D3 treatment, and 1 d after the treatment p21Cip1/WAF1 was induced in the nucleus. However, it was mainly localized in the cytoplasm after 3 d, a time point when the monocytic differentiation was well evident. As we noticed that young neurons which express ß-tubulin display cytoplasmic p21Cip1/WAF1 expression, whereas the precursor cells not expressing ß-tubulin did not (Fig. 1 B), it is suggested that cytoplasmic p21Cip1/WAF1 is induced during the developmental stage after differentiation in neurons as well as monocytic cells and has relevant roles.
Zhou et al. (2001) previously reported a possible mechanism of translocation of p21Cip1/WAF1, which was triggered by Akt-induced phosphorylation of p21Cip1/WAF1 at residues Thr145. As Thr145 is in the NLS of p21Cip1/WAF1, phosphorylation of p21Cip1/WAF1 may result in the loss of its nuclear localization ability. However, another group casts doubt on this finding, as they could not confirm translocation of p21Cip1/WAF1 by this phosphorylation (Rossig et al., 2001). More investigation will be required to address this discrepancy; therefore, we did not use the constitutive phosphorylated mutant of p21Cip1/WAF1 in our study. Truncation of the nuclear localization signal is also the mechanism of regulation of subcellular localization of p21Cip1/WAF1. At an early phase during DNA damage-induced apoptosis, the COOH-terminal of p21Cip1/WAF1 is truncated by a member of the caspase family of proteases (Gervais et al., 1998; Levkau et al., 1998; Zhang et al., 1999), and after cleavage p21Cip1/WAF1 loses its NLS and exits from the nucleus (Levkau et al., 1998). The NLS-p21 construct we used here was similar to this truncated p21Cip1/WAF1 and worked well in our system. However, as we observed the signals for GFP-
NLS-p21 also in the nucleus of transfected cells and the hippocampal neurons, GFP-
NLS-p21 would enter the nucleus by passive diffusion (Lang et al., 1986).
Cytoplasmic p21Cip1/WAF1 inhibits Rho-kinase activity
Rho-kinase plays important roles in, for example, stress fiber, and focal adhesion formation (Leung et al., 1996; Amano et al., 1997), smooth muscle contraction (Kureishi et al., 1997), cytokinesis (Yasui et al., 1998), and neurite retraction (Amano et al., 1998), as a downstream effector of Rho (Matsui et al., 1996). Some chemical compounds have been shown to inhibit Rho-kinase activity (Uehata et al., 1997). Staurosporine, HA1077 and Y-32885 inhibited the activity of Rho-kinase as well as protein kinase N, one of the targets of Rho, and the IC50 values of these toward Rho-kinase were 7 nM, 1.7 µM, and 0.4 µM, respectively (Amano et al., 1999). In this study, p21Cip1/WAF1 inhibited Rho-kinase activity in a dose-dependent manner, and the IC50 value was 1.43 nM, suggesting the strong inhibitory effect.
Rho/Rho-kinase and the neurite outgrowth
A number of factors that regulate Rho activity are implicated in neurite outgrowth and growth cone guidance (for review see Luo, 2000). We showed previously that the axonal outgrowth was facilitated by the ligand binding to the neurotrophin receptor p75 presumably through inactivation of Rho (Yamashita et al., 1999). In addition, our observation that myelin-associated glycoprotein as well as tumor necrosis factor elicited inhibition of neurite outgrowth and branching seems to be mediated by the activation of Rho (Neumann et al., 2002; Yamashita et al., 2002). Taking these findings into consideration, blocking the activity of Rho-kinase would be a good molecular target, as the axonal outgrowth should be promoted by blocking the downstream pathway even if Rho is activated by some cytokines or guidance molecules. In fact, Rho-kinase was shown to be a possible therapeutic target for central nervous system axon regeneration (Lehmann et al., 1999).
However, not all the neuronal cells respond to various stimuli in the same way. In PC12 cells during differentiation after nerve growth factor treatment, ectopic expression of constitutively active Rho does not cause the disappearance of neurites (Sebok et al., 1999). In dorsal root ganglion neurons up to postnatal day 3 to 4 or embryonic retinal ganglion neurons, axonal outgrowth was not significantly inhibited by myelin-associated glycoprotein, which activates Rho (Johnson et al., 1989; Mukhopadhyay et al., 1994; De Bellard et al., 1996; Cai et al., 2001; Yamashita et al., 2002). Although these reports suggest that the responses of the neurons to Rho depend on the cell context, another interpretation of the data is that immature or young neurons may have intrinsic mechanisms to overcome the inhibitory effects mediated by Rho. The molecular mechanisms that govern these phenomenon remain to be elucidated, however, our notion that cytoplasmic p21Cip1/WAF1 promotes neurite outgrowth through inactivation of Rho-kinase may be an interesting hypothesis to explain the loss of responses to Rho activation. Future studies will address these issues.
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Materials and methods |
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Plasmid constructs
pEGFP-full-p21 (aa 1164) and pEGFP-NLS-p21 (aa 1140) are mammalian expression vectors for GFP fused proteins (Asada et al., 1999). Myc-Rho-kinase in pEF-BOS was provided by Dr. K. Kaibuchi (Nagoya University, Nagoya, Japan).
Cell culture and transfection
NIH3T3 cells, N1E-115 cells, and 293T cells were maintained in DME containing 10% fetal bovine serum. Lipofectamine 2000 (Invitrogen) was used for transfection. For the stress fiber formation assay, NIH3T3 cells were cultured in serum-free medium for 16 h after transfection. Stress fiber formation was evoked by incubating the cells with 10% serum for 10 min. Hippocampal neurons were prepared from 18-d-old Sprague-Dawley rats, as previously described (Neumann et al., 1995). Briefly, hippocampi were dissected and the meninges removed. The trimmed tissue was dissociated by trituration. The dissociated cells were plated on dishes precoated with poly-L-lysine (Sigma-Aldrich), and cultured in DME containing 10% fetal bovine serum for 24 h. Then, the medium was replaced with DME with B27 supplement (Invitrogen), and the cells were transfected with GFP or GFP-NLS-p21. Neuronal morphology was estimated at 24 h after the transfection.
Morphological analysis of N1E-115 cells
N1E-115 cells were transfected with GFP, GFP-full-p21, or GFP-NLS-p21, and cultured in serum-starved condition for 5 h. Then, the medium was replaced with DME containing 10% fetal bovine serum. The cells were fixed at 48 h after transfection. The morphology of the cells was categorized into three groups; neurite-positive cells, round cells, and the other cells. The cells with longer neurites than their soma were defined as neurite positive cells. The other cells had various features including microspikes, ruffles and a flattened appearance.
Coimmunoprecipitation of NLS-p21 and Rho-kinase
293T cells were transfected with myc-Rho-kinase in combination with GFP-full-p21 or GFP-NLS-p21. At 48 h after transfection, the cells were lysed with 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Nonidet-P40 including protease inhibitor cocktail tablets; Roche). The cell lysates were centrifuged at 13,000 g for 20 min, and the supernatant was collected. Immunoprecipitations were performed for 2 h at 4°C using an anti-p21Cip1/WAF1 mouse monoclonal antibody (Santa Cruz Biotechnology) and 0.75 ml of the supernatant. The immunocomplexes were collected with protein G-Sepharose (Amersham Pharmacia Biotech) slurry (50% vol/vol), washed four times with lysis buffer, and subjected to SDS-PAGE. They were transferred to the polyvinylidene difluoride membranes and probed with the anti-myc rabbit polyclonal antibody (Santa Cruz Biotechnology). Interaction of endogenous proteins in N1E-115 cells was assessed in the same way using antiRho-kinase antibody.
In vitro binding assay
Recombinant full-length p21Cip1/WAF1 (1164, >98% purity, 1 nM; Santa Cruz Biotechnology) and purified GST fused protein of a fragment of Rho-kinase (GST-CAT; aa 6553) were incubated in 1 ml of the buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 1 mM EDTA including protease inhibitor cocktail tablets) for 2 h, and GST-CAT was precipitated using glutathione sepharose (Amersham Pharmacia Biotech). The resultant precipitates were electrophoretically transferred to polyvinylidene difluoride membranes after SDS/PAGE with 10% gels and were immunoblotted with the anti-p21Cip1/WAF1 antibody.
Kinase assay
The kinase reaction for Rho-kinase was carried out using a S6 Kinase Assay Kit (Upstate Biotechnology) according to the manufacturer's instructions. Briefly, for in vitro assay, 10 µl of assay dilution buffer (ADB: 20 mM MOPS, pH 7.2, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate and 1 mM dithiothreitol), 10 µl of substrate cocktail (250 µM substrate peptide [AKRRRLSSLRA] in ADB), 10 µl of the inhibitor cocktail, 10 µl of the [-32P] ATP mixture (magnesium/ATP cocktail including 10 µCi of the [
-32P] ATP) and 20 mU of Rho kinase fragment (aa 1543; Upstate Biotechnology) were mixed. After incubation with p21Cip1/WAF1 protein for 10 min at 30°C, the reaction mixtures were spotted onto the P81 phosphocellulose paper and quantified using a scintillation counter.
For the in vivo assay, 293T cells were cotransfected with myc-Rho-kinase in combination with GFP or p21Cip1/WAF1 constructs. Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet-P40 and protease inhibitor cocktail). The kinase assay was carried out using the lysates.
Immunostaining
For immunohistochemistry, sections of chick retinas were permeabilized and blocked with the blocking buffer (0.1% Triton X-100, 0.1% BSA, and 5% goat serum in PBS) for 30 min at room temperature. For immunocytochemistry, cells were permeabilized and blocked with the buffer containing 0.2% Triton X-100. They were incubated overnight at 4°C with the anti-p21Cip1/WAF1 antibody (1:1000) and an antiß-tubulin class III rabbit polyclonal antibody (TuJ1) (1:2,000; Research Diagnostic, Inc.), followed by incubation for 1 hour with Alexa 488-labeled goat antimouse IgG antibody (Molecular Probes) and Alexa 568-labeled goat antirabbit IgG antibody (Molecular Probes). Tetramethyl rhodamine isothiocyanate-labeled phalloidin (1:1,000; Sigma-Aldrich) was used to detect F-actin in NIH3T3 cells and N1E-115 cells. Hippocampal neurons were immunostained with the anti-TuJ1 antibody. When necessary, DAPI (300 nM; Wako) was used to stain the nucleus. Samples were examined under a confocal laser-scanning microscope (Carl Zeiss).
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Footnotes |
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Acknowledgments |
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Submitted: 15 February 2002
Revised: 30 May 2002
Accepted: 30 May 2002
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References |
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