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J Biol Chem, Vol. 275, Issue 13, 9805-9813, March 31, 2000

Apoptosis Signal-regulating Kinase 1 (ASK1) Induces Neuronal Differentiation and Survival of PC12 Cells^*

Kohsuke Takeda, Takiko Hatai, Tatsuo S. Hamazaki, Hideki Nishitoh, Masao Saitoh, and Hidenori Ichijo

From the Department of Biomaterials Science, Faculty of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1N) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1N in PC12 cells. ASK1N-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1N induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1N-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The rat pheochromocytoma cell line PC12 is a useful model of neuronal differentiation and death (1). On treatment with nerve growth factor (NGF),¹ PC12 cells differentiate with sympathetic neuron-like characteristics including neurite outgrowth. Upon binding of NGF, the cell surface receptor tyrosine kinase is activated, and the activated kinase in turn activates the small GTP-binding protein Ras followed by sequential phosphorylation and activation of three members of the mitogen-activated protein kinase (MAPK) superfamily, MAPK kinase kinase (MAPKKK)/Raf, MAPK kinase (MAPKK)/MEK, and MAPK/extracellular signal-regulated kinase (ERK) (2-4). Activation of the ERK pathway is thought to play roles during NGF-induced neuronal differentiation, since constitutively active MEK induced neurite outgrowth and selective blockade of the ERK pathway using dominant-negative MEK or the MEK inhibitor PD98059 resulted in inhibition of NGF-induced neurite outgrowth (5-7). It has also been suggested that persistence of ERK activation is critical for differentiation of PC12 cells, since transient activation of ERK induced by agents such as EGF is insufficient to induce neurite outgrowth (8-10).

However, a contradictory finding was recently reported, in which an interfering mutant of the small G protein Rap1 which blocks the sustained phase of ERK activation did not inhibit NGF-induced neurite outgrowth, suggesting that sustained activation of ERK is not necessarily required for NGF-induced neuronal differentiation (11). In another study, NGF-induced neurite outgrowth of PC12D cells, a subline of PC12 cells, was shown to be resistant to the addition of PD98059 (12). Furthermore, neuronal differentiation of PC12 cells by treatment with bone morphogenetic protein (BMP)-2 was induced in the absence of ERK activation (13). These studies strongly suggest that signaling pathways other than the ERK cascade also contribute to neuronal differentiation of PC12 cells.

Candidates for signaling pathways regulating neuronal cell death and differentiation include two different recently identified MAPK cascades that converge on c-Jun N-terminal kinase (JNK; also known as SAPK, stress-activated protein kinase) and p38 MAP kinase. Whereas the ERK signaling cascade is generally involved in the control of cell proliferation and differentiation, JNK and p38 are preferentially activated by cytotoxic stressors such as UV radiation, x-rays, heat shock and osmotic shock, and by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (14-16). Importantly, activation of the JNK and/or p38 pathway has been observed in response to deprivation of trophic factors in differentiated PC12 cells and other neuronal cells, suggesting the possible involvement of JNK and p38 in neuronal death (17-20). When the JNK pathway was inhibited using a dominant-interfering c-Jun mutant, neuronal death elicited by trophic factor withdrawal was decreased, implying that the JNK pathway is a necessary component of neuronal death induced by trophic factor deprivation. Moreover, mice lacking the JNK3 gene, a member of the JNK family (21), and JunAA mice, in which endogenous Jun is replaced by a dominant-negative mutant Jun allele (22), were reported to exhibit marked reduction in excitotoxicity-induced apoptosis of hippocampal neurons, clearly demonstrating the requirement of JNK activity for neuronal death. More recently, compound mutant mice lacking the JNK1 and JNK2 genes were shown to be embryonic lethal and have severe dysregulation of apoptosis in brain, suggesting that JNK1 and JNK2 regulate region-specific apoptosis during early brain development (23).

On the other hand, several lines of evidence have suggested that the JNK and p38 pathways are involved in neuronal differentiation. Differentiation of PC12 cells induced by expression of GTPase-deficient, constitutively active forms of the heterotrimeric G_q family members, G_q and G₁₆, was accompanied by persistent activation of JNK but not of ERK (24). Expression of constitutively activated c-Jun, which partly mimicked the phosphorylated form of the protein, or retrovirus-mediated overexpression of c-Jun induced neuronal differentiation of PC12 cells independently of upstream signals (25). Staurosporine, a protein kinase inhibitor and promoter of neurite outgrowth in PC12 cells, specifically induced prolonged activation of a novel JNK isoform (26). In addition, p38 was recently shown to be activated in response to NGF and to be required for NGF-induced differentiation of PC12 cells (27, 28). More recently, it was reported that p38 was activated by treatment with BMP-2 in PC12 cells and that activation of p38 might be sufficient to induce neuronal differentiation of PC12 cells (29). These observations suggested that the JNK and p38 pathways mediate important biological signals not only for neuronal cell death but also for neuronal differentiation.

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed MAPKKK that activates the SEK1-JNK and MKK3/MKK6-p38 signaling cascades (30). Overexpression of ASK1 in epithelial cells in low serum condition induced apoptosis, and in ovarian cancer cells expression of a kinase-inactive mutant of ASK1 inhibited microtubule-interfering agent-induced apoptosis, suggesting that ASK1 plays a role in the mechanism of stress-induced apoptosis (30-32). We recently found that overexpression of ASK1 induced death of NGF-differentiated PC12 cells and primary rat sympathetic neurons (SCGs). Moreover, dominant-negative ASK1 reduced the neuronal death induced by NGF withdrawal from these cells (33). ASK1 was activated upon treatment with TNF- or agonistic anti-Fas antibody, and a kinase-inactive mutant of ASK1 reduced TNF-- and Fas-induced JNK activation and apoptosis, suggesting that ASK1 is a pivotal component in cytokine-induced apoptosis as well (30, 34, 35). Considering the involvement of JNK and p38 in neuronal death and differentiation as described above, it is of great interest whether ASK1 mediates signals leading to death or differentiation of undifferentiated PC12 cells.

We report here that transient or stable expression of constitutively active ASK1 (ASK1N) induces neurite outgrowth in PC12 cells. We found that p38 and to a lesser extent JNK, but not ERK, were activated by expression of ASK1N in PC12 cells. Experiments using MEK inhibitors and a p38 inhibitor suggested a significant contribution of activation of p38, rather than of ERK, to the neurite-inducing activity of ASK1 in PC12 cells. We also found that ASK1N induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by expression of ASK1N. Moreover, we found that ASK1N-expressing PC12 cells survived in serum-starved condition. Taken together, these findings suggest that ASK1 may mediate signals leading to both differentiation and survival of undifferentiated PC12 cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Wild-type (WT)-PC12 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 10% heat-inactivated horse serum (HS), and 100 units/ml penicillin G in a 5% CO₂ atmosphere at 37 °C.

Antibodies and Reagents-- Rat monoclonal antibody to the hemagglutinin (HA) tag (clone 3F10) was purchased from Roche Molecular Biochemicals. Anti-SAPK/JNK polyclonal antibody, anti-ERK polyclonal antibody, and phospho-specific polyclonal antibodies to p38 MAP kinase (Thr-180/Tyr-182) and SAPK/JNK (Thr-183/Tyr-185) were purchased from New England Biolabs. Phospho-specific polyclonal antibody to ERK (Thr-183/Tyr-185) was purchased from Promega. Anti-p38 MAP kinase goat polyclonal antibody (C-20-G), anti-neurofilament medium subunits (NF-M) polyclonal antibody, and anti-Tau monoclonal antibody were purchased from Santa Cruz Biotechnology Inc., Affiniti Research Products Ltd., and Transduction Laboratories, respectively. Monoclonal antibody to neurofilament heavy subunits (NF-H) clone RMO-24, which specifically recognizes a phosphate-dependent epitope in the tail domain of NF-H (36, 37), was purchased from Zymed Laboratories Inc.. Anti-NF-H monoclonal antibody clone N52, which recognizes phosphorylated and non-phosphorylated NF-H, and anti--tubulin isotype III monoclonal antibody were purchased from Sigma.

SB203580, PD98059, and U0126 were purchased from Calbiochem, New England Biolabs, and Promega, respectively. Mouse 2.5S NGF was purchased from Takara.

Adenovirus Vectors-- Recombinant adenoviruses encoding HA-tagged ASK1 mutants and -galactosidase were constructed as described (31). Nearly 100% infection of recombinant adenoviruses to PC12 cells can be achieved at a multiplicity of infection (m.o.i.) of 100 as determined by -galactosidase staining (data not shown).

Tetracycline (Tet)-regulated Expression System-- The hygromycin resistance gene derived from pBHMr and the neomycin resistance gene derived from pABWN were each subcloned into pTet-tTAk plasmid (Life Technologies, Inc.) and pTet-Splice (Life Technologies, Inc.) and named pTet-tTAk-hyg and pTet-Splice-neo, respectively. The cDNA for HA-tagged ASK1N (HA-ASK1N) was then subcloned into pTet-Splice-neo. The pTet-tTAk-hyg plasmid was transfected into PC12 cells using DMRIE-C (Life Technologies, Inc.) according to the manufacturer's instruction. The cells were then selected with 240 units/ml hygromycin B (Wako), and several hygromycin-resistant clones were established. A clone termed PC12-tTA was further transfected with pTet-Splice-neo-based expression vector for HA-ASK1N. After selection with 400 µg/ml neomycin (Geneticin, Life Technologies, Inc.) and 240 units/ml hygromycin, the double-resistant clones were established and analyzed for the expression of HA-ASK1N proteins by Western blot. The cells were maintained in DMEM supplemented with 10% FCS, 10% HS, 100 units/ml penicillin G, 500 ng/ml Tet (Sigma), 120 units/ml hygromycin, and 200 µg/ml neomycin.

Neurite Outgrowth Assay-- Approximately 1 × 10⁵ WT-PC12 cells per well were plated in 6-well cell culture plates (Becton Dickinson Labware) coated with bovine type IV collagen (Cellmatrix, Nitta Gelatin) and allowed to grow in DMEM supplemented with 10% FCS and 10% HS for overnight. Cells were then washed twice with phosphate-buffered saline (PBS) and refed with DMEM containing 1% HS and a recombinant adenovirus encoding HA-ASK1N or -galactosidase. Twenty-four hours after infection, cells were washed with PBS and further cultured in DMEM containing 1% HS for an additional 48 h. In the case of PC12-ASK1N cells, approximately 1 × 10⁵ cells per well were plated in type IV collagen-coated 6-well cell culture plates and allowed to grow in DMEM supplemented with 10% FCS, 10% HS, and 500 ng/ml Tet overnight. Cells were then washed twice with PBS and refed with DMEM containing 1% HS and given concentrations of Tet. To determine the percentage of neurite-bearing cells, the number of cells with a process longer than one cell diameter was determined and compared with the total number of cells counted.

Western Blot Analysis-- Cells were lysed in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS, and cell extracts were clarified by centrifugation. To detect the expression of neuronal marker proteins, cells were lysed in the same lysis buffer except with the concentration of SDS increased to 1% and sonicated briefly before clarification by centrifugation. The protein concentration of the supernatant was measured using a DC Protein Assay (Bio-Rad), and the same amounts of protein were resolved on SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes. After blocking with 5% skim milk in TBS-T (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.05% Tween 20) for 1 h, the membranes were probed with antibodies. The antibody-antigen complexes were detected using the ECL system (Amersham Pharmacia Biotech).

Survival Assay-- Approximately 5 × 10⁴ PC12-ASK1N cells per well were plated in type IV collagen-coated 24-well cell culture plates and allowed to grow in DMEM supplemented with 10% FCS, 10% HS, and 500 ng/ml Tet overnight. The cells were washed twice with PBS and refed with serum-free DMEM containing given concentrations of Tet (day 0). Using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay-based Cell Counting Kit-8 (Dojindo), relative cell numbers were determined in triplicate by estimating the value of day 0 as 1.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Constitutively Active ASK1 Induces Neurite Outgrowth in PC12 Cells-- To investigate the roles played by ASK1 in neuronal cells, we examined the effect of expression of ASK1N, a constitutively active mutant of ASK1 (31), in PC12 cells. First, wild-type (WT)-PC12 cells were infected with recombinant adenoviruses encoding hemagglutinin (HA) epitope-tagged ASK1N (Ad-ASK1N) or -galactosidase (Ad--galactosidase) in culture medium containing 1% horse serum (HS). Nearly 100% infection of recombinant adenoviruses to WT-PC12 cells was achieved at an m.o.i. of 100 as determined by -galactosidase staining (data not shown). With Western blot analysis using a monoclonal anti-HA antibody, expression of ASK1N was detected in an m.o.i.-dependent manner 24 h after infection (Fig. 1A). Unexpectedly, at this time point, Ad-ASK1N-infected cells began to sprout neurites (data not shown). To confirm the neurite-inducing activity of ASK1, WT-PC12 cells left uninfected or infected with Ad-ASK1N were cultured for 72 h and examined by phase-contrast microscopy. The cells infected with Ad-ASK1N (m.o.i. of 100) exhibited marked extension of neurites (Fig. 1B, right panel), whereas uninfected cells exhibited no neurite extension (Fig. 1B, left panel). When cells with a neurite longer than one cell diameter were counted, the percentage of neurite-bearing cells increased by Ad-ASK1N infection in an m.o.i.-dependent manner (Fig. 1C). Taken together with the observation that infection with Ad-ASK1KM, a recombinant adenovirus encoding a kinase-inactive mutant of ASK1 (31), or Ad--galactosidase failed to induce neurite outgrowth in PC12 cells (data not shown), these findings indicate that the neurite outgrowth induced by infection with Ad-ASK1N is dependent on the kinase activity of ASK1.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Constitutively active ASK1 (ASK1N)-induced neurite outgrowth in WT-PC12 cells. A, adenovirus-mediated expression of ASK1N in WT-PC12 cells. WT-PC12 cells were infected with the indicated m.o.i. of a recombinant adenovirus encoding HA-tagged ASK1N (Ad-ASK1N). Twenty-four h after the infection, cells were lysed and immunoblotted with a monoclonal antibody to HA. B, induction of neurite outgrowth in WT-PC12 cells. WT-PC12 cells were infected with an m.o.i. 100 of Ad-ASK1N in DMEM containing 1% HS. Twenty-four h after the infection, cells were washed with PBS and further cultured in fresh medium for an additional 48 h. Morphology of uninfected cells (left panel) and Ad-ASK1N-infected cells (right panel) was examined by phase-contrast microscopy. Bar, 50 µm. C, dose-dependent effect of Ad-ASK1N on neurite outgrowth in WT-PC12 cells. WT-PC12 cells were infected with the indicated m.o.i. of Ad-ASK1N in DMEM containing 1% HS. Twenty-four h after infection, cells were washed with PBS and further cultured in fresh medium for an additional 48 h. The percentage of cells with a process longer than one cell diameter was determined. Results are the means of three independent experiments ± S.E.

To examine further the roles of ASK1 in PC12 cells, we established a stable cell line expressing HA-tagged ASK1N (PC12-ASK1N cells), in which the expression of ASK1N is under the control of a Tet-repressible promoter (see "Experimental Procedures"). After the complete removal of Tet from the culture medium, expression of ASK1N was turned on and first detectable as early as 3 h later by Western blot analysis (data not shown). Twenty-four h after the reduction of Tet in the culture medium, no leaky expression of ASK1N was detected in the presence of Tet (500 ng/ml), and the level of ASK1N expression was tightly controlled by the concentration of Tet (Fig. 2A). Fig. 2B shows representative morphology of PC12-ASK1N cells expressing (Tet 0 ng/ml) or not expressing (Tet 500 ng/ml) ASK1N 24 h after the reduction of Tet. The ASK1N-expressing cells exhibited marked extension of neurites, similar to WT-PC12 cells infected with Ad-ASK1N.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Induction of neurite outgrowth in PC12 cells stably transfected with HA-ASK1N (PC12-ASK1N cells). A, expression of ASK1N in PC12-ASK1N cells. PC12-ASK1N cells, stably expressing HA-tagged ASK1N under the control of a Tet-repressible promoter, were cultured in DMEM containing 1% HS and the indicated concentration of Tet for 24 h. Cells were then lysed and immunoblotted with a monoclonal antibody to HA. B, induction of neurite outgrowth in PC12-ASK1N cells. PC12-ASK1N cells were cultured in DMEM containing 1% HS in the presence (left panel) or absence (right panel) of Tet. Twenty-four h after the reduction of Tet, cell morphology was examined by phase-contrast microscopy. Bar, 50 µm. C, time course quantification of neurite-bearing PC12-ASK1N cells at different concentrations of Tet. PC12-ASK1N cells were cultured in DMEM containing 1% HS in the presence of the indicated concentration of Tet. The percentage of cells with a process longer than one cell diameter was determined at different time points after the reduction of Tet. Results are the means of three independent experiments.

We next examined the time and concentration effects of Tet on the induction of neurite outgrowth in PC12-ASK1N cells. Twenty-four h after the reduction of Tet, approximately 15% of cells extended neurites with 50 ng/ml Tet (Fig. 2C). With 0 or 10 ng/ml of Tet, the proportion of neurite-bearing cells increased to approximately 45-50% in parallel with the expression of ASK1N protein detected by Western blot (Fig. 2, A and C). Forty eight h after the reduction of Tet, the proportion of neurite-bearing cells further increased in a time-dependent manner with 50 or 10 ng/ml Tet. However, when Tet was completely removed from the culture medium (Tet 0 ng/ml), the proportion of neurite-bearing cells peaked at 24 h but was decreased at 48 and 72 h. With a longer period of culture (up to 2 weeks), ASK1N-induced neurite outgrowth was found to be most prominent at 10 ng/ml Tet (data not shown).

ASK1N Preferentially Activates the p38 MAP Kinase Pathway in PC12 Cells-- ASK1 activates the JNK and p38 MAP kinase pathways in COS cells (30). To explore the signaling pathway(s) leading to the induction of neurite outgrowth by ASK1, we examined which MAP kinase cascade is activated in PC12 cells by the expression of ASK1N. To measure the activation status of endogenous ERK, JNK, and p38, we performed Western blot analysis using polyclonal antibodies that specifically recognize the dually phosphorylated active forms of these enzymes. We tested the dose-dependent effect of ASK1N expression on activation of MAPKs in PC12-ASK1N cells 24 h after the reduction of Tet (Fig. 3A). Consistent with our previous report for COS cells (30), an intense activation of p38 was readily detected with expression of ASK1N. When the same membrane was reprobed with anti-phospho-JNK antibody, intense signals corresponding to phosphorylated p54 and p46 isoforms of JNK were detected in the lysate of sorbitol-treated cells, indicating the presence of intact signaling components leading to JNK in this cell line. Unexpectedly, in contrast to p38, JNK was barely activated by ASK1N; only marginal activation of JNK was detected when an excess amount of ASK1N was expressed at 10 or 0 ng/ml Tet. We detected no clear increase in ERK activation induced by ASK1N on reprobing the same membrane with the anti-phospho-ERK antibody (data not shown; see below).

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Effect of ASK1N expression on p38 and JNK in PC12 cells. A, preferential activation of p38 by ASK1. PC12-ASK1N cells were cultured in DMEM containing 1% HS and the indicated concentration of Tet for 24 h. As positive controls for activation of JNK and p38, cells were exposed to 0.5 M sorbitol for 5 min in the presence of 500 ng/ml Tet. Cells were then lysed and immunoblotted with specific antibodies to HA, phosphorylated p38, total p38, phosphorylated JNK, and total JNK. B, time course of activation of p38 and JNK with different concentrations of Tet in PC12-ASK1N cells. PC12-ASK1N cells were cultured in DMEM containing 1% HS and the indicated concentration of Tet for 24, 48, and 72 h. Cells were lysed at each time point and immunoblotted with specific antibodies as indicated. As a positive control, the lysate of the cells exposed to 0.5 M sorbitol for 5 min in the presence of 500 ng/ml Tet was used (indicated as S). C, activation of p38 in WT-PC12 cells by infection with Ad-ASK1N. WT-PC12 cells were infected with an m.o.i. 50 (+) or 200 (++) of Ad-ASK1N or an m.o.i. 100 (+) or 400 (++) of Ad-ASK1KM for 24 h. As a positive control, cells were exposed to 0.5 M sorbitol for 5 min. Cells were then lysed and immunoblotted with specific antibodies to HA, phosphorylated p38, and total p38.

We next examined the time course effects of ASK1N on JNK and p38 in PC12-ASK1N cells (Fig. 3B). In this experiment, we used the same sets of Tet concentration and time points as those in Fig. 2C, in order to determine the correlation between the ASK1N-induced neurite outgrowth and the activation of downstream MAPKs. With 50 ng/ml Tet, expression of ASK1N increased time-dependently and reached a plateau at 48 h, and constant activation of p38 was maintained from 24 to 72 h. On the other hand, activation of JNK was first detectable 72 h after reduction of Tet. Neither p38 nor JNK was activated in the cells maintained in the presence of Tet (500 ng/ml) throughout the time course of observation. At 10 or 0 ng/ml Tet, activation of p38 peaked at 24 h and decreased thereafter in accordance with the expression level of ASK1N, whereas clear activation of JNK was first detected 48 h after reduction of Tet. The preferential activation of p38 by ASK1N was further confirmed in WT-PC12 cells, which we infected with Ad-ASK1N or Ad-ASK1KM and tested for activation of MAPKs 24 h after infection. Although there was a quantitative difference in the adenovirus-mediated expression of ASK1N and ASK1KM, expression of ASK1N, but not of ASK1KM, clearly activated p38 but not JNK or ERK (Fig. 3C and data not shown). These observations strongly suggest that ASK1N preferentially activates the p38 pathway in PC12 cells. Furthermore, together with the observation that ASK1N-induced neurite outgrowth was already found within 24 h after the induction of ASK1N, it is likely that p38, rather than JNK or ERK, plays a primary role in ASK1N-induced neurite outgrowth in PC12 cells.

Basal Activity of ERK Only Minimally Contributes to ASK1N-induced Neurite Outgrowth-- It has been suggested that activation of the ERK pathway plays crucial roles in NGF-induced neuronal differentiation. Since we did not detect ASK1N-dependent activation of ERK in either WT-PC12 or PC12-ASK1N cells, we examined whether basal activity of ERK is required for ASK1N-induced neurite outgrowth (Fig. 4). In PC12-ASK1N cells, a relatively high level of basal phosphorylation of ERK was observed in the presence of 500 ng/ml Tet, but no additional phosphorylation was induced by expression of ASK1N. In the presence of 10 mM each of the MEK inhibitors PD98059 and U0126, the phosphorylation of ERK was partially and completely inhibited by PD98059 and U0126, respectively. When the percentage of neurite-bearing cells was determined 48 h after the induction of ASK1N in the presence or absence of the MEK inhibitors, PD98059 and U0126 were found to only slightly decrease the number of neurite-bearing cells. Importantly, despite the obvious difference in ERK phosphorylation status, no significant difference was detected between PD98059 and U0126 in ability to modulate ASK1N-induced neurite outgrowth. These results suggest that the ERK pathway makes little contribution, if any, to ASK1N-induced neurite outgrowth in PC12 cells.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Effect of MEK inhibitors on ASK1N-induced neurite outgrowth in PC12-ASK1N cells. PC12-ASK1N cells were plated in duplicate in 6-well cell culture plates and cultured in DMEM containing 1% HS and 500 or 10 ng/ml Tet in the presence or absence of 10 µM MEK inhibitors. The MEK inhibitors, PD98059 and U0126, are indicated as PD and U, respectively. Cells in one plate were lysed 24 h after the reduction of Tet and immunoblotted with specific antibodies to phosphorylated ERK, total ERK, and HA. The cells in another plate were used for quantification of neurite outgrowth 48 h after reduction of Tet. The percentage of cells with a process longer than one cell diameter was determined. Results are the means of three independent experiments ± S.E.

SB203580 Inhibits ASK1N-induced Neurite Outgrowth-- To assess the requirement of p38 for ASK1N-induced neurite outgrowth, we examined the effect of SB203580, a specific inhibitor of p38, on the neurite-inducing activity of ASK1N. When PC12-ASK1N cells were induced to extend neurites in the presence or absence of SB203580, ASK1N-induced neurite outgrowth was inhibited in the presence of only 200 nM SB203580 (Fig. 5A). We confirmed that this concentration of SB203580 had no cytotoxic effect on PC12-ASK1N cells (data not shown). Fig. 5B shows that the neurite-inducing activity of ASK1N was inhibited in a dose-dependent manner by addition of SB203580. Since the presence of higher concentrations above 200 nM SB203580 in the culture medium decreased the expression of ASK1N, we could not determine whether higher concentrations of SB203580 completely inhibited ASK1N-induced neurite outgrowth. However, these findings strongly suggest that p38 is required for the neurite outgrowth induced by ASK1N.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Effect of SB203580 on ASK1N-induced neurite outgrowth in PC12-ASK1N cells. A, morphological examination of the effect of SB 203580 on ASK1N-induced neurite outgrowth. PC12-ASK1N cells were cultured in DMEM containing 1% HS and 10 ng/ml Tet in the presence (right panel) or absence (left panel) of SB203580 for 48 h. Cell morphology was examined by phase-contrast microscopy. Bar, 50 µm. B, dose-dependent effect of SB203580 on ASK1N-induced neurite outgrowth. PC12-ASK1N cells were cultured in DMEM containing 1% HS and 10 ng/ml Tet in the presence of different concentrations of SB203580 for 48 h. The percentage of cells with a process longer than one cell diameter was determined. Results are the means of three independent experiments ± S.E.

Characterization of ASK1-induced Differentiation of PC12 Cells-- To confirm that the neurite outgrowth induced by ASK1N was accompanied by neuronal differentiation of PC12 cells, we examined the expression of several neuron-specific proteins as markers of neuronal differentiation. For comparison with NGF-induced differentiation, PC12-ASK1N cells were cultured in parallel in the presence of 50 ng/ml NGF and 500 ng/ml Tet. PC12-tTA cells, the parental cell line of PC12-ASK1N cells that stably expresses only the gene for a tetracycline transactivator (see "Experimental Procedures"), were also cultured under the same conditions. After culture for 10 days with or without induction of ASK1N, cells were lysed and subjected to Western blot analysis using a variety of antibodies to neuron-specific markers (Fig. 6A). -Tubulin isoform III (tubulin-III) is synthesized exclusively by neurons of higher vertebrates and increases in conjunction with the rate of neuronal differentiation (38). By using a monoclonal antibody to tubulin-III, we detected a marked increase in the expression of tubulin-III following treatment with NGF in both PC12-ASK1N and PC12-tTA cells. PC12-ASK1N cells but not PC12-tTA cells cultured in the reduced concentration of Tet also exhibited a dramatic increase in the expression of tubulin-III, indicating that the up-regulation of tubulin-III was induced by the expression of ASK1N and was not a nonspecific effect of Tet reduction from the culture medium. Similar results were achieved using a monoclonal antibody to Tau, which is a group of microtubule-associated proteins (MAPs), and is known to be selectively expressed in unique subsets of neurons (39). Consistent with a previous report (40), expression of Tau was up-regulated in both cells by treatment with NGF. Expression of ASK1N also increased Tau protein in PC12-ASK1N cells. These results indicated that ASK1N-induced morphological change of PC12-ASK1N cells was accompanied by up-regulation of neuron-specific components of microtubules, as was the case with NGF-induced differentiation.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Effect of ASK1N expression on neuron-specific proteins in PC12 cells. A, effect of ASK1N expression on neuron-specific proteins in PC12-ASK1N and PC12-tTA cells. PC12-ASK1N and PC12-tTA cells were cultured in DMEM containing 1% HS and 500 (+) or 10 ng/ml () Tet in the presence (+) or absence () of 50 ng/ml NGF for 10 days. Cells were then lysed and immunoblotted with specific antibodies to phosphorylated NF-H, total NF-H, NF-M, Tau, tubulin-III, and HA. B, effect of ASK1N expression on neuron-specific proteins in WT-PC12 cells. WT-PC12 cells were infected with Ad-ASK1N or Ad--galactosidase (Ad--gal) in the presence (+) or absence () of 50 ng/ml NGF in DMEM containing 1% HS. Twenty-four h after the infection, the cells were washed with PBS and further cultured in fresh medium for an additional 48 h. Cells were lysed and immunoblotted with specific antibodies to phosphorylated NF-H, total NF-H, and HA. C, time course of phosphorylation of NF-H induced by expression of ASK1N in PC12-ASK1N cells. PC12-ASK1N cells were cultured in DMEM containing 1% HS in the presence (+) or absence () of Tet. Cells were lysed at the indicated time points and immunoblotted with antibodies to phospho-NF-H, total NF-H, HA, phospho-p38, and total p38.

We next examined the expression of neurofilament proteins, which are major elements of the neuronal cytoskeleton. Neurofilaments are composed of three intermediate filament proteins, neurofilament light (NF-L), neurofilament medium (NF-M), and neurofilament heavy (NF-H). Of these three subunits, NF-M and NF-H have extensive C-terminal domains including major phosphorylation sites (41). In untreated PC12-ASK1N and PC12-tTA cells, a polyclonal antibody to NF-M detected endogenous NF-M as a doublet band. Since this antibody recognizes NF-M independently of its phosphorylation state, the observed slower and faster migrated bands were thought to correspond to phosphorylated and non-phosphorylated forms of NF-M, respectively. In both cells, treatment with NGF significantly increased both the phosphorylated and non-phosphorylated forms of NF-M. Interestingly, expression of ASK1N in PC12-ASK1N cells induced a band shift of NF-M without a change in total amount, suggesting that ASK1N induces phosphorylation of NF-M in vivo. A monoclonal antibody that recognizes NF-H independently of its phosphorylation state detected endogenous NF-H as a single band in untreated PC12-ASK1N and PC12-tTA cells. When the cells were treated with NGF, expression of NF-H was detected as a broad band, suggesting that NGF up-regulates both protein level and phosphorylation of NF-H. Notably, expression of NF-H in the ASK1N-expressing cells was also detected as a broad band with extremely slowly migrating species, suggesting that expression of ASK1N strongly induces phosphorylation of NF-H. To confirm the ASK1N-induced phosphorylation of NF-H, we next used a monoclonal antibody that specifically recognizes highly phosphorylated forms of NF-H (36, 37). The anti-phospho-NF-H antibody clearly detected the highly phosphorylated NF-H in the lysate of the ASK1N-expressing cells, suggesting again that ASK1N and/or its downstream kinase(s) phosphorylates neurofilaments.

To examine whether the adenovirus-mediated expression of ASK1N also induces phosphorylation of NF-H, we infected WT-PC12 cells with Ad-ASK1N or Ad--galactosidase and lysed them 72 h after infection. The cell lysate was subjected to Western blot analysis using anti-NF-H and anti-phospho-NF-H antibodies (Fig. 6B). The anti-NF-H antibody, but not the anti-phospho-NF-H antibody, detected basal expression of NF-H as a single band in untreated Ad--galactosidase-infected cells. When the Ad--galactosidase-infected cells were treated with NGF, expression of NF-H was detected as a slightly broad band, suggesting that 72 h treatment with NGF induces phosphorylation of NF-H to some extent in WT-PC12 cells. Consistent with the results for PC12-ASK1N cells, NF-H expressed in Ad-ASK1N-infected cells was detected as a doublet band, the upper band of which was recognized by the anti-phospho-NF-H antibody, demonstrating that adenovirus-mediated expression of ASK1N strongly induced phosphorylation of NF-H in WT-PC12 cells.

To investigate further the kinetics of phosphorylation of NF-H induced by ASK1, we examined the ASK1N-induced phosphorylation of NF-H over a short time course, i.e. within 24 h. In PC12-ASK1N cells, phosphorylation of NF-H was first detected 12 h after the removal of Tet, following the expression of ASK1N and the activation of p38, which were readily detected 8 h after Tet removal (Fig. 6C). Since up-regulation of other neuron-specific proteins required several days of culture after the induction of ASK1N (data not shown), phosphorylation of NF-H was a nearly immediate event caused by expression of ASK1N. These results suggest that p38 activated by ASK1 or ASK1 itself directly phosphorylates NF-H.

Taken together, these findings indicate that ASK1N-induced morphological change of PC12 cells was accompanied by the expression of neuron-specific proteins and intense phosphorylation of neurofilament proteins. These findings strongly suggest that ASK1N-induced neurite outgrowth results from neuronal differentiation of PC12 cells.

ASK1N-expressing PC12 Cells Survive in Serum-starved Condition-- NGF induces not only differentiation but also survival of neurons. To test the possibility that ASK1 also mediates survival signals in PC12 cells, we tested the effect of ASK1N expression on serum-starved PC12-ASK1N cells. PC12-ASK1N cells were cultured in different concentrations of Tet in serum-free medium for up to 7 days. Relative number of surviving cells was determined at different time points after the reduction of serum and Tet (Fig. 7A). In contrast to the gradual decrease in the number of ASK1N-non-expressing cells (Tet 500 ng/ml), the number of ASK1N-expressing cells (50 or 0 ng/ml of Tet) was maintained or even increased in the serum-free medium for 7 days, suggesting that ASK1N-expressing cells were viable in serum-starved condition. It also appeared that moderate expression of ASK1N is required to obtain the optimal survival effect, since this effect was more prominent in 50 ng/ml than 0 ng/ml Tet. Fig. 7B shows the morphology of the cells expressing (Tet 50 ng/ml) or not expressing (Tet 500 ng/ml) ASK1N cultured in the serum-free medium for 5 days. ASK1N-expressing cells in the serum-free medium appeared similar to those in the medium containing 1% HS (Fig. 2B), whereas the cells not expressing ASK1N had an apoptotic appearance and detached from the culture plate. We also found that PC12-tTA and WT-PC12 cells infected with Ad-ASK1N, but not with Ad-ASK1KM or Ad--galactosidase, survived in the serum-free medium (data not shown). These observations suggest that moderate activation of ASK1 is capable of mediating survival signal in PC12 cells. ASK1 thus appeared to mediate signals leading to both differentiation and survival of PC12 cells.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Effect of ASK1N expression on serum-starved PC12-ASK1N cells. A, time course quantification of surviving PC12-ASK1N cells. Using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay-based Cell Counting Kit-8 (Dojindo), the relative number of surviving cells in serum-free DMEM containing the indicated concentration of Tet was determined at different time points after reduction of serum and Tet, which was performed on day 0. Results are the means of triplicate determinations ± S.E. from a representative experiment. B, morphological examination of the effect of ASK1N expression on serum-starved PC12-ASK1N cells. PC12-ASK1N cells were cultured in serum-free DMEM containing the indicated concentration of Tet. Morphology of the cells expressing (right panel) and not expressing (left panel) ASK1N was examined by phase-contrast microscopy 5 days after serum withdrawal. Bar, 50 µm.

SB203580 Inhibits ASK1N-mediated Survival Effect-- We next assessed the requirement of p38 for the ASK1N-mediated survival effect by treatment with SB203580. To avoid evaluating the inhibitory effect of SB203580 on ASK1N-induced differentiation, we used PC12-ASK1N cells differentiated by expressing ASK1N under optimal culture conditions (in DMEM containing 10 ng/ml of Tet and 1% HS) for 1 week. The differentiated PC12-ASK1N cells were then cultured in the presence (50 or 200 nM) or absence of SB203580 in serum-free medium containing 50 ng/ml Tet, the concentration optimal for ASK1N-mediated survival effect (Fig. 7A). At different time points after the deprivation of serum, the relative number of surviving cells was determined (Fig. 8). In the absence of SB203580, the same number of differentiated PC12-ASK1N cells was maintained in the serum-free medium for 3 days, confirming that ASK1N-expressing cells were viable in serum-starved condition. In the presence of SB203580, however, the number of surviving cells was decreased, indicating that SB203580 inhibited the survival effect mediated by ASK1N. These findings suggest that activation of p38 is required not only for ASK1N-induced neurite outgrowth but also for ASK1N-mediated survival effect and that p38 is an important downstream mediator of ASK1 signaling in PC12 cells.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effect of SB203580 on serum-starved PC12-ASK1N cells. PC12-ASK1N cells were differentiated in DMEM containing 10 ng/ml Tet and 1% HS for 7 days and then cultured in the presence (50 or 200 nM) or absence (containing the equivalent volume of dimethyl sulfoxide (DMSO)) of SB203580 in serum-free medium containing 50 ng/ml Tet for an additional 3 days. By using a Cell Counting Kit-8, the relative number of surviving cells was determined at different time points after the reduction of serum. Results are the means of triplicate determinations ± S.E. from a representative experiment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we demonstrated that expression of ASK1N induced neuronal differentiation of undifferentiated PC12 cells, which was characterized by neurite outgrowth, induction of neuron-specific proteins, and phosphorylation of neurofilament proteins. In addition, ASK1 appeared to transduce survival signal in PC12 cells.

Concomitant with the marked neurite outgrowth, rapid and intense activation of p38 was induced by expression of ASK1N (Fig. 3). In contrast, no additional activation of ERK was induced by expression of ASK1N, and inhibition of basal activity of ERK did not markedly decrease the number of ASK1N-induced neurite-bearing cells (Fig. 4), suggesting that ASK1N-induced neurite outgrowth does not require the activation of ERK. Given that NGF-induced differentiation of PC12 cells requires activation of the ERK pathway and is sensitive to MEK inhibitors (7, 10), we suggest that a major set of signaling pathways mediating the neurite-inducing activity of ASK1 differs from that activated in NGF-induced differentiation of PC12 cells. Expression of a dominant-negative mutant form of ASK1 (ASK1KM) consistently did not inhibit the NGF-induced neurite extension in PC12 cells (data not shown). Importantly, a p38 inhibitor inhibited ASK1N-induced neurite outgrowth (Fig. 5), suggesting that the activation of p38 is required for this morphological event. Recently, p38 was reported to be required for NGF- and BMP-2-induced differentiation of PC12 cells (28, 29). Although it is still unknown whether activation of p38 is sufficient to induce neurite outgrowth and/or neuron-specific markers, p38 appears to be a generally required element in neuronal differentiation of PC12 cells.

We previously reported that ASK1 activates both the JNK and p38 pathways in various cell types including COS cells, SCGs, and NGF-differentiated PC12 cells (30, 33). Unexpectedly, ASK1-induced activation of JNK was relatively weak in undifferentiated PC12 cells. Whereas intense activation of p38 was easily detected within 24 h after the induction of ASK1N, activation of JNK was first detected only after 48 h (Fig. 3B). Since the neurite outgrowth was induced within 24 h after the induction of ASK1N, it is unlikely that the activation of JNK triggers neuronal differentiation of PC12 cells. JNK and its substrate c-Jun have been suggested to participate in death signaling of neurons, especially trophic factor deprivation-induced death. Interestingly, it was recently reported that expression of constitutively active MEKK1 (MEKK1) in PC12 cells induced activation of both JNK and p38 to a similar extent, and that these cells underwent apoptosis (20), and that a dominant-interfering c-Jun mutant could inhibit MEKK1-induced apoptosis, suggesting the possible involvement of JNK in death signaling in undifferentiated PC12 cells. Our observation that expression of ASK1N induced differentiation, but not death, in PC12 cells may be related to the absence of intense activation of JNK. In addition, expression of ASK1N in PC12-ASK1N cells with the complete absence of Tet (0 ng/ml), but not with the moderate reduction of Tet (50 or 10 ng/ml), resulted in a decrease in the number of neurite-bearing cells after a longer period of culture (Fig. 2C). The differential effect of ASK1 may be explained by the relative increase in JNK activity in the complete absence of Tet (Fig. 3B). Although the extent of JNK activation induced by ASK1N is insufficient to elicit death of PC12 cells, the weak activation of JNK by ASK1 might antagonize the neurite-inducing activity of the ASK1-p38 axis. It may also be possible that optimal balance between activation of the JNK and p38 pathways is required for ASK1N-induced neurite outgrowth in PC12 cells.

We found that expression of ASK1N up-regulated neuron-specific components of microtubules and strongly induced phosphorylation of neurofilament proteins (Fig. 6), suggesting that neurite outgrowth induced by expression of ASK1N indeed resulted from neuronal differentiation of PC12 cells. Surprisingly, the extent of phosphorylation of NF-H induced by expression of ASK1N was much more prominent than that induced by treatment with NGF. Phosphorylation of NF-H is thus a major characteristic of ASK1N-induced differentiation of PC12 cells. NF-M and NF-H are among the most highly phosphorylated proteins in the nervous system. Although the role of their phosphorylation is not fully understood, phosphorylated NF-M and NF-H are major components of the cytoskeleton in many types of mature neurons and are particularly abundant in myelinated axons (41). Most of the phosphorylation sites in NF-M and NF-H are located in the C-terminal tail region containing multiple copies of Lys-Ser-Pro (KSP) motif (42, 43). To date, several kinases including cyclin-dependent kinase 5 (Cdk5) (44), glycogen synthase kinase-3 (GSK-3) (45), ERK1 and -2 (46, 47), and JNK1 (48, 49) have been implicated in the phosphorylation of NF-M and NF-H. Relevant to the present study, it is of interest that proline-directed MAP kinases ERK and JNK are reported to phosphorylate directly NF-H (47, 48), since p38, another proline-directed kinase, may phosphorylate NF-H in ASK1N-expressing PC12 cells. Together with the observation that the phosphorylation of NF-H was detectable soon after the activation of p38 (Fig. 6C), it is possible that p38, rather than JNK, activated by ASK1 directly phosphorylates NF-H. Alternatively, other unidentified kinase(s) activated by ASK1 or ASK1 itself may phosphorylate NF-H.

The observation that ASK1N-expressing cells could survive in serum-starved condition suggests another unexpected function of ASK1 (Fig. 7). We found that expression of ASK1N induced apoptosis in NGF-differentiated PC12 cells and SCGs (33). Although these findings appear to contradict those of the present study, we suggest that the expression level of ASK1N could determine the cell fate; moderate expression of ASK1N induces differentiation and survival, whereas excessive ASK1 activity induces apoptosis in PC12 cells. In fact, we observed that excess overexpression of ASK1N induced apoptosis even in undifferentiated PC12 cells (data not shown). On the other hand, it is difficult to compare the precise expression level of transfected ASK1N in undifferentiated PC12 cells with that in NGF-differentiated PC12 cells or SCGs; we also consider the possibility that ASK1 may preferentially mediate apoptosis signal in NGF-differentiated PC12 cells and SCGs due to the different cellular context. We are currently investigating the difference in the ASK1-induced activation of downstream signaling pathways between undifferentiated and NGF-differentiated PC12 cells.

Similar to the ASK1N-induced neurite outgrowth, ASK1N-mediated survival effect was inhibited by a p38 inhibitor (Fig. 8), suggesting that the activation of p38 plays crucial roles in both differentiation and survival induced by ASK1. However, in the absence of data in primary neurons, we cannot exclude a possibility that the differentiation/pro-survival effects of ASK1 might be a PC12-specific phenomenon. Future experiments using undifferentiated neuronal precursor cells may enable understanding of diverse roles of ASK1 in neuronal physiology.

	ACKNOWLEDGEMENTS

We thank K. Miyazono, Y. Sahara, K. Tobiume, Y. Sawada, A. Matsuzawa, Y. Mochida, S. Inoshita, K. Morita, and T. Kanamoto for valuable discussions.

	FOOTNOTES

^* This work was supported by CREST of Japan Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biomaterials Science, Faculty of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Tel.: +81-3-5803-5471; Fax: +81-3-5803-0192; E-mail: takeda.det2@dent.tmd.ac.jp (K. T.), ichijo.det2@dent.tmd.ac.jp (H. I.).

	ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; ASK1, apoptosis signal-regulating kinase 1; ERK, extracellular signal-regulating kinase; HS, horse serum; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated kinase; SCG, superior cervical ganglia; Tet, tetracycline; MAP, mitogen-activated protein; MEK, MAPK/ERK kinase; SAPK, stress-activated protein kinase; BMP, bone morphogenetic protein; TNF, tumor necrosis factor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HA, hemagglutinin; PBS, phosphate-buffered saline; WT, wild type; m.o.i., multiplicity of infection; tubulin-III, -tubulin isoform III; Ad, adenovirus; NF-L, neurofilament light; NF-M, neurofilament medium; NF-H, neurofilament heavy; hyg, hygromycin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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