Communicated by:Kunihiro Matsumoto
Dorsomorphin stimulates neurite outgrowth in PC12 cells via activation of a protein kinase A-dependent MEK-ERK1/2 signaling pathway
Article first published online: 12 OCT 2011
© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd
Genes to Cells
Volume 16, Issue 11, pages 1121–1132, November 2011
How to Cite
Kudo, T.-a., Kanetaka, H., Mizuno, K., Ryu, Y., Miyamoto, Y., Nunome, S., Zhang, Y., Kano, M., Shimizu, Y. and Hayashi, H. (2011), Dorsomorphin stimulates neurite outgrowth in PC12 cells via activation of a protein kinase A-dependent MEK-ERK1/2 signaling pathway. Genes to Cells, 16: 1121–1132. doi: 10.1111/j.1365-2443.2011.01556.x
- Issue published online: 23 OCT 2011
- Article first published online: 12 OCT 2011
- Received: 25 February 2011 Accepted: 29 August 2011
In this study, we investigated the effect of dorsomorphin, a selective inhibitor of bone morphogenetic protein (BMP) signaling, on rat PC12 pheochromocytoma cell differentiation. PC12 cells can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor, BMP2, and other inducers. Cells were incubated with BMP2 and/or dorsomorphin, and the extent of neurite outgrowth was evaluated. Unexpectedly, BMP2-mediated neuritogenesis was not inhibited by co-treatment with dorsomorphin. We also found that treatment with dorsomorphin alone, but not another BMP signaling inhibitor, LDN-193189, induced neurite outgrowth in PC12 cells. To further understand the mechanism of action of dorsomorphin, the effects of this drug on intracellular signaling were investigated using the following signaling inhibitors: the ERK kinase (MEK) inhibitor U0126; the tropomyosin-related kinase A inhibitor GW441756; and the protein kinase A (PKA) inhibitor H89. Dorsomorphin induced rapid and sustained ERK1/2 activation; however, dorsomorphin-mediated ERK1/2 activation and neuritogenesis were robustly inhibited in the presence of U0126 or H89, but not GW441756. These findings suggest that dorsomorphin has the potential to induce neuritogenesis in PC12 cells, a response that requires the activation of PKA-dependent MEK-ERK1/2 signaling.
PC12 is a cloned rat cell line isolated from an adrenal medullary pheochromocytoma and an excellent model for studying biomolecular events involved in neuronal differentiation and the effects of various chemicals on neurite outgrowth (Vaudry et al. 2002; Radio & Mundy 2008). PC12 cells are induced to differentiate into neuron-like cells by various factors, including nerve growth factor (NGF), fibroblast growth factors, born morphogenetic proteins (BMPs) and cyclic adenosine monophosphate (cAMP) (Greene & Tischler 1976; Rydel & Greene 1987; Deutsch & Sun 1992; Lazarovici et al. 1998; Koike et al. 2006; Evangelopoulos et al. 2009). A number of signaling molecules have been reported to be activated by inducers of neuritogenesis in PC12 cells (Lazarovici et al. 1998; Tsuji et al. 2001; Yanagisawa et al. 2001; Vaudry et al. 2002; Charles et al. 2003; Obara et al. 2004; Yasui et al. 2010).
The addition of NGF to PC12 cells causes the rapid and sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated protein kinase (MAPK) family, through the tropomyosin-related kinase A (TrkA)-mediated activation of the small monomeric G-protein Ras and Ras-associated protein-1 (Rap1). However, sustained activation is dependent on the activation of ERK1/2 by Rap1 (Marshall 1998; Rakhit et al. 2001; Vaudry et al. 2002). Sustained activation leads to neurite outgrowth and the development of many of the phenotypic characteristics of PC12 cells (Greene & Tischler 1976; Kao et al. 2001; Rakhit et al. 2001). The NGF-mediated differentiation of PC12 cells also requires the participation of p38 MAPKs. A recent study showed that the inhibition of NGF-induced p38 MAPK activation blocked neurite outgrowth in PC12 cells, although the activation of ERK1/2 induced by NGF was essential for NGF-mediated p38 MAPK phosphorylation (Yung et al. 2008). However, the overall mechanisms that link the activation of NGF receptors to neurite growth are not well defined.
Similar to NGF, cAMP can induce neuritogenesis in neuronal cells and PC12 cells (Vossler et al. 1997; Freeland et al. 2000; Yang et al. 2008). However, the cAMP signaling pathway that activates neuritogenesis in PC12 cells is distinct from the pathway activated by NGF (Frödin et al. 1994; Vaudry et al. 2002; Obara et al. 2004; Yang et al. 2008). The elevation of cAMP levels in PC12 cells causes the sustained TrkA- and Ras-independent activation of ERK1/2, whereas the activation of ERK1/2 occurs through protein kinase A (PKA)-dependent Rap1-B-raf-ERK1/2 kinase (MEK) signaling (Yao et al. 1998; Grewal et al. 2000; Hansen et al. 2003; Obara et al. 2004).
Bone morphogenetic proteins are members of the large transforming growth factor-β (TGF-β) cytokine superfamily (Wrana et al. 1994) that interact with two classes of transmembrane receptor serine–threonine protein kinases, termed type I and type II receptors (Hogan 1996), and activate two downstream pathways, the Smad signaling pathway and the TGF-β-associated kinase 1 (TAK1)-p38 MAPK signaling pathway (Heldin et al. 1997; Kimura et al. 2000). BMP2 causes neurite outgrowth in PC12 cells in a different way from that induced by NGF (Iwasaki et al. 1996). In the case of the BMP-induced neuronal differentiation of PC12 cells, neurite outgrowth is dependent on BMP-mediated TAK1-p38 MAPK signaling and is totally independent of ERK1/2 signaling (Iwasaki et al. 1999; Yanagisawa et al. 2001).
Because the low-molecular-weight compound dorsomorphin (Fig. 1), also known as compound C (Zhou et al. 2001), was recently identified as a selective inhibitor of BMP type I receptors and can be used to inhibit the BMP signaling pathway (Hao et al. 2008; Yu et al. 2008b; Kim et al. 2010), we first sought to clarify the effect of dorsomorphin on the BMP2-mediated neuronal differentiation of PC12 cells. For this purpose, we analyzed neurite outgrowth in the presence of BMP2 and/or dorsomorphin during the course of neuronal differentiation in PC12 cells. We unexpectedly found that dorsomorphin induced neurite outgrowth independently in PC12 cells. Here, we report the characterization of dorsomorphin-induced neurite outgrowth in PC12 cells and discuss a possible mechanism of dorsomorphin action.
Induction of neurite outgrowth in dorsomorphin-treated PC12 cells
To examine the effects of the selective BMP signaling inhibitor dorsomorphin (Fig. 1) on the neuronal differentiation of PC12 cells, we first investigated the effects of dorsomorphin on BMP2-induced neurite outgrowth. PC12 cells were incubated in the presence or absence of 40 ng/mL BMP2 and/or 2 μm dorsomorphin for 2 days, and the extent of neurite outgrowth was evaluated on day 2. Before drug treatment on day 0, the cells were relatively small and round with few visible neurites (Fig. 2A); the extent of neurite outgrowth was <3% in PC12 cells incubated with no additives for 2 days (Fig. 2B,F). Treatment with BMP2, which is an inducer of neuritogenesis in PC12 cells (Iwasaki et al. 1996), resulted in the gradual induction of neurite outgrowth by day 2 (Fig. 2C,F), although BMP2-induced neurite outgrowth in the cells was not strong, as previously reported (Hayashi et al. 2001). Unexpectedly, no suppression of neurite outgrowth was observed on day 2 in PC12 cells treated with 40 ng/mL BMP2 in the presence of 2 μm dorsomorphin. Rather, neuritogenesis was enhanced as a result of co-treatment with these drugs (Fig. 2D,F), despite a previous report on the inhibitory effect of dorsomorphin on BMP signaling (Yu et al. 2008b). We also found that treatment with 2 μm dorsomorphin alone significantly induced the outgrowth of neurite-like processes in PC12 cells in the absence of BMP2 co-treatment, similar to our results with BMP2 (40 ng/mL) and dorsomorphin (2 μm) (Fig. 2E,F). The morphology of the neurite-like processes formed as a result of dorsomorphin treatment resembled that of the BMP2-induced neurites (see Fig. 2C).
Dose response and time course of dorsomorphin-induced neurite outgrowth in PC12 cells
We showed that dorsomorphin alone can induce neurite outgrowth in PC12 cells, similar to BMP2; thus, we next evaluated the dose response and time course of dorsomorphin-dependent neurite outgrowth in PC12 cells. We first scored PC12 cells for neurite outgrowth after a 2-day incubation with various concentrations of dorsomorphin (0–2 μm) or another BMP signaling inhibitor, LDN-193189 (0–2 μm). PC12 cells were also scored for neurite outgrowth at the indicated times (0–48 h) after the addition of 2 μm dorsomorphin. As shown in Fig. 3A,B, dorsomorphin-mediated neurite outgrowth occurred rapidly in a dose- and time-dependent manner. However, LDN-193189 did not induce neuritogenesis at all, despite the fact that its chemical structure is similar to that of dorsomorphin (Figs 1 and 3C,D). These results imply that dorsomorphin has the potential to induce neuritogenesis via an unknown and original mechanism that differs from the BMP2-dependent mechanism and that the dorsomorphin-dependent mechanism of neuritogenesis may involve the rapid activation of an intracellular signaling pathway.
Rapid and sustained activation of ERK1/2 in dorsomorphin-treated PC12 cells
Activation of the ERK1/2 signaling pathway induces neuritogenesis in PC12 cells (Kao et al. 2001). ERK1/2 activation and neurite outgrowth induced by NGF are inhibited by the MEK1/2 inhibitors PD098059 and U0126 (Pang et al. 1995; Ahn & Tolwinski 1999). Thus, we next assessed whether dorsomorphin activates the ERK1/2 signaling pathway. For this purpose, cell lysates were analyzed for endogenous ERK1/2 activity at 15, 60 and 240 min after the addition of 2 μm dorsomorphin to cell cultures in the presence or absence of 5 μm U0126. Next, we evaluated the time course of ERK1/2 activity by Western blotting after the addition of dorsomorphin to the cell culture. As shown in Fig. 4A–C, the significant induction of ERK1/2 activity peaked 15 min after dorsomorphin was added and sustained ERK1/2 activation was observed. The dorsomorphin-dependent activation of ERK1/2 was completely abolished in the presence of U0126 (Fig. 4A–C). No significant change in endogenous ERK1/2 expression was observed after the addition of dorsomorphin (Fig. 4A).
As treatment with 2 μm dorsomorphin induced the immediate activation of ERK1/2 signaling and peak ERK1/2 activity was detected 15 min after drug treatment (Fig. 4A–C), we next evaluated the dose responsiveness of dorsomorphin-dependent ERK1/2 activation in PC12 cells during that time period. For this purpose, PC12 cells were treated with various concentrations of dorsomorphin (0–2 μm) for 15 min, and cell lysates were analyzed for endogenous ERK1/2 activity by Western blotting using antiphosphorylated (activated) ERK1/2 antibodies. As shown in Fig. 4D,E, the dorsomorphin-mediated activation (phosphorylation) of ERK1/2 increased in a dose-dependent manner in PC12 cells.
Suppression of dorsomorphin-induced neurite outgrowth by U0126 in PC12 cells
The results presented in Fig. 4 prompted us to determine whether dorsomorphin-mediated activation of the ERK1/2 signaling pathway is required for dorsomorphin-mediated neurite outgrowth in PC12 cells. We therefore pretreated cells with the MEK1/2 inhibitor U0126 at a concentration high enough to abolish dorsomorphin-induced ERK1/2 activation (5 μm) (Fig. 4A–C) before the 2-day induction of dorsomorphin-dependent neurite outgrowth. The results presented in Fig. 5A–C show that 5 μm U0126 almost completely suppressed dorsomorphin-induced neuritogenesis in PC12 cells. We also scored PC12 cells incubated in the presence of U0126 at various concentrations (0–5 μm) to assess the inhibition of neurite outgrowth. Our results indicate that U0126 reduced dorsomorphin-induced neuritogenesis in PC12 cells in a dose-dependent manner (Fig. 5D). These results suggest that dorsomorphin-induced neurite outgrowth in PC12 cells requires the sustained activation of MEK-ERK1/2 signaling.
Suppression of dorsomorphin-induced neurite outgrowth in PC12 cells by H89, but not GW441756
To further elucidate the signaling pathways involved in dorsomorphin-dependent neuritogenesis in PC12 cells, we next attempted to clarify whether TrkA and PKA, possible upstream kinases in the ERK1/2 signaling pathway in PC12 cells, are involved. For this purpose, dorsomorphin-mediated neurite outgrowth was induced in PC12 cells in the presence or absence of the TrkA kinase-specific inhibitor GW441759, the PKA-specific inhibitor H89 and U0126 (as a control). PC12 cells were scored for neurite outgrowth after incubation for 2 days. The results presented in Fig. 6A–F show that H89, at a concentration high enough to inhibit PKA activation (5 μm) (Hansen et al. 2003), suppressed dorsomorphin-induced neuritogenesis in PC12 cells, similar to treatment with 5 μm U0126. In contrast, GW441756, at a concentration high enough to suppress TrkA activation (1 μm) (Jung et al. 2008), had no significant effect on neurite outgrowth. These results suggest that PKA signaling, but not TrkA signaling, is required for dorsomorphin-induced neuritogenesis in PC12 cells.
Inhibition of the dorsomorphin-induced activation of ERK1/2 in PC12 cells by H89
We finally examined whether the inhibition of PKA activity also affects dorsomorphin-induced ERK1/2 activation in PC12 cells. As peak ERK1/2 activity was detected 15 min after treatment with the chemical at 2 μm (Fig. 4A), we evaluated dorsomorphin-dependent ERK1/2 activation in PC12 cells during that time period in the presence or absence of H89 (0–5 μm). Lysates from these cells were analyzed for endogenous ERK1/2 activity by Western blotting using antiphosphorylated (activated) ERK1/2 antibodies. As shown in Fig. 7A–C, the dorsomorphin-induced activation (phosphorylation) of ERK1/2 in the cells was reduced in a dose-dependent manner by H89. These results suggest that dorsomorphin-induced MEK-ERK1/2 signaling during dorsomorphin-dependent neurite outgrowth in PC12 cells requires PKA activity.
In this study, we found that the low-molecular-weight compound dorsomorphin increased the phosphorylation of endogenous ERK1/2 and induced neuritogenesis in PC12 cells, whereas the structurally related BMP signaling inhibitor LDN-193189 had no effect on neurite outgrowth (Figs 2–4). Treatment with the MEK1/2-specific inhibitor U0126 or the PKA-specific inhibitor H89 attenuated dorsomorphin-induced neuritogenesis considerably (Figs 5 and 7). These data indicate the essential role of PKA-mediated MEK-ERK1/2 signaling in the dorsomorphin-dependent neuronal induction of PC12 cells. In this context, it is reasonable to assume that the effects of dorsomorphin on ERK1/2 activation and neurite outgrowth in PC12 cells showed in this study were not attributable to the dorsomorphin solvent, diethyl sulfoxide (DMSO), but to dorsomorphin itself, because: (i) it was previously shown that DMSO does not induce ERK1/2 activation or neurite outgrowth, at least at concentrations of up to 0.2% (v/v), which is the maximum concentration of DMSO used in this study (Figs 2–7) (Tamura & Ohkuma 1991; Ahn & Tolwinski 1999; Shinomiya & Shinomiya 2003; Radio et al. 2008; Lin et al. 2010); and (ii) both of the BMP signaling inhibitors used in this study (dorsomorphin and LDN-193189) were also dissolved in the same solvent (DMSO) (Fig. 3). Our data also show that dorsomorphin itself does not directly activate ERK1/2, because the inhibition of MEK1/2 by U0126 totally abolished dorsomorphin-mediated ERK1/2 activation (Fig. 4).
It is still unclear how dorsomorphin induces PKA-mediated MEK-ERK1/2 activation and subsequent neuritogenesis in PC12 cells. Our data showed that the TrkA tyrosine kinase inhibitor GW441756 had no effect on dorsomorphin-induced neurite outgrowth in PC12 cells (Fig. 6), suggesting that the activation of PKA-mediated MEK-ERK1/2 signaling is independent of TrkA-mediated signaling. Because (i) dorsomorphin rapidly activated ERK1/2 in the absence of BMP2 in PC12 cells (Fig. 4) and (ii) it has already been shown that dorsomorphin inhibits other kinases such as AMP-activated kinase (AMPK) and receptor tyrosine kinases for PDGF and VEGF, in addition to BMP type I receptors (Yu et al. 2008b; Hao et al. 2010), dorsomorphin may increase ERK1/2 activity by affecting molecular target(s) other than BMP type I receptors that are closely involved in the activation of a PKA-dependent MEK-ERK1/2 signaling cascade. Because the cAMP-dependent kinase PKA is the main mediator of cAMP signaling, and intracellular cAMP levels are regulated by the activity of adenylate cyclase (Taylor et al. 1990), there is significant value in investigating whether dorsomorphin regulates the generation of cAMP by adenylate cyclase in PC12 cells through an unknown mechanism. In support of this hypothesis, cAMP has already been shown to induce neuritogenesis in PC12 cells via PKA (see Introduction for details).
Alternatively, the dorsomorphin-mediated inhibition of basal BMP signaling may indirectly activate PKA-mediated MEK-ERK1/2 signaling. However, we clearly showed in the present study that another BMP signaling inhibitor, LDN-193189, which presents much greater specificity for BMP type I receptors as a kinase inhibitor (Cuny et al. 2008; Yu et al. 2008a; Boergermann et al. 2010; Hao et al. 2010), did not induce neurite outgrowth by itself in PC12 cells (Fig. 3). Therefore, the latter possibility described above may not be very likely.
We also showed sustained ERK1/2 activation in dorsomorphin-treated PC12 cells (Fig. 4). The data collected in this study are in basic agreement with the previous finding that the sustained activation of ERK1/2 exerted a marked influence on neuronal differentiation in PC12 cells (Burry 2001; Yasui et al. 2010). ERK1/2 activation is dependent on at least two distinct small G-proteins, Ras and Rap1, which link growth factor receptors to the MEK-ERK1/2 signaling cascade by activating the MEK kinases c-Raf (also known as Raf-1) and B-Raf, respectively (Segal et al. 1996; Marshall 1998). Therefore, these features of the ERK1/2 activation mechanism will help us to identify the signaling components that link PKA to MEK-ERK1/2 signaling. This will enable us to clarify the mechanism of action of dorsomorphin in PC12 cells in future studies.
Interestingly, PKA signaling can either stimulate or inhibit MEK-ERK1/2 signaling depending on the cell type and cellular conditions (Stork & Schmitt 2002; Obara et al. 2004). In mouse fibroblast NIH 3T3 cells, rat fibroblast Rat-1 cells and mouse pluripotent mesenchymal precursor C2C12 cells, PKA indirectly suppresses ERK1/2 activity through the inhibition of c-Raf or activation of the ERK1/2-specific protein phosphatase MAPK phosphatase-1 (MKP-1). On the other hand, PKA signaling activates ERK1/2 in other cell types, including human prostate carcinoma LNCaP cells, rat pituitary GH4C1 cells and rat neuronal PC12 cells (Stork & Schmitt 2002; Ghayor et al. 2009). Consistent with these studies, recent work has shown that dorsomorphin does not independently enhance ERK1/2 activity in C2C12 cells (Boergermann et al. 2010), suggesting that the rapid and sustained PKA-mediated activation of ERK1/2 in response to dorsomorphin may not occur in all mammalian cells but may instead occur in a cell type- or cell content-dependent manner.
Neuronal differentiation is a highly complex process that must be precisely orchestrated through a number of signaling pathways. Defects in neuronal differentiation may result in various neurological disorders, including neurodegenerative diseases (Yung et al. 2008). Although a variety of methods can be used to analyze developmental pathways, small molecules have the advantage of being able to penetrate cells more easily than peptides and of being more stable than them (Kim et al. 2010). Thus, our data support the idea that the selective small molecule dorsomorphin and perhaps its derivatives may become highly valuable tools for investigating and regulating the complex process of neuronal differentiation.
In conclusion, we characterized the novel action of dorsomorphin in the differentiation of PC12 cells. We have clearly shown that dorsomorphin induces neuritogenesis through PKA-dependent MEK-ERK1/2 activation, rather than through activation of the NGF receptor TrkA. Further investigation into the mechanisms underlying dorsomorphin action and the identification of its critical target(s) related to neuritogenesis in PC12 cells will be highly valuable and will expand our understanding of the regulation of neuronal differentiation.
Recombinant human BMP2 obtained from Peprotech (Rocky Hill, NJ, USA) was dissolved in LF6 buffer solution (5 mm glutamic acid, 5 mm NaCl, 2.5% glycine, 0.5% sucrose, and 0.01% Tween 80). Dorsomorphin, a selective inhibitor of BMP type I receptors (Hao et al. 2008; Yu et al. 2008b), was obtained from Calbiochem (San Diego, CA, USA). The chemical structure of this compound is shown in Fig. 1. Another BMP signaling inhibitor, LDN-193189 (see Fig. 3C; Cuny et al. 2008), was purchased from Stemgent (San Diego, CA, USA). The MEK1/2-specific inhibitor U0126 and the PKA-specific inhibitor H89 were obtained from Calbiochem. The TrkA-specific tyrosine kinase inhibitor GW441756 was purchased from Axon Medchem BV (Groningen, The Netherlands). Each signaling inhibitor used in the present study was dissolved in DMSO, which was obtained from Wako Pure Chemical Industries (Osaka, Japan). Antiphosphorylated (activated) ERK1/2 antibodies, anti-ERK1/2 antibodies and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA).
Cell culture and the induction of differentiation
Rat pheochromocytoma PC12 cells were provided by RIKEN BRC (Tsukuba, Japan) through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 5% horse serum (Life Technologies) and penicillin/streptomycin under a 5% CO2 atmosphere at 37 °C. For the neurite outgrowth assays, cells were seeded in growth medium at 2 × 104 or 6 × 104 cells/well in collagen type IV-coated, 6-well and 24-well culture plates (BD Biosciences, Bedford, MA, USA), respectively, and allowed to grow for 24 h. Thereafter, the cells were cultured under serum-starved conditions (DMEM supplemented with 1% horse serum), and BMP2 and/or dorsomorphin was added. Neurite outgrowth was quantified by examining the cells using phase-contrast microscopy (DP72; Olympus Corp., Tokyo, Japan). Three random photographs were taken per well, and cells bearing processes 1.5 times longer than the length of the cell body were considered positive. A total of at least 300 cells were counted per well; each data point corresponds to the counts obtained from 3 independent wells. When cells were treated with a signaling inhibitor, the drug was added to the cultures before addition of the neuritogenesis inducer. The final concentration of DMSO used in this study as the solvent for dorsomorphin was a maximum of 0.2% (v/v), which did not induce neurite outgrowth in PC12 cells (data not shown), as reported previously (Tamura & Ohkuma 1991; Shinomiya & Shinomiya 2003; Radio et al. 2008).
Cells were lysed in ice-cold lysis buffer [50 mm Tris–HCl (pH 8.0), 150 mm NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.2 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA)]. The resulting cell lysates were centrifuged at 13 750 g for 5 min at 4 °C. The supernatants were used as cell extracts. The protein extract concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equivalent amounts of protein extract were subjected to SDS–PAGE and electroblotted onto polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Piscataway, NJ, USA). The membranes were blocked with 1% (w/v) blocking reagent (Roche Diagnostics) in Tris-buffered saline containing Tween 20 [TBST: 20 mm Tris–HCl (pH 7.5), 137 mm NaCl and 0.1% (v/v) Tween 20] for 2 h at room temperature. After an overnight incubation with antiphosphorylated (activated) ERK1/2 antibodies (1 : 3000) or anti-ERK1/2 antibodies (1 : 1500), the membranes were washed four times with TBST over a 20-min period and incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (1 : 1000) for 30 min. After four washes with TBST, the signals were seen using the Enhanced Chemiluminescence (ECL) Plus Western Blotting Detection System (GE Healthcare) with an ImageQuant LAS4000 mini imaging system (GE Healthcare). The intensity of the bands was determined using ImageQuant TL software (GE Healthcare).
The data are presented as the mean ± SEM. Significant differences between groups were identified by one-way analysis of variance followed by Holm’s testing. P-values <0.05 were considered statistically significant.
The authors thank Mr Tsuyoshi Kan-no, Ms Kanako Tominami and Ms Ayako Okumoto for providing technical assistance.
- 1999) U0126: an inhibitor of MKK/ERK signal transduction in mammalian cells. Promega Notes 71, 4–8. & (
- 2010) Dorsomorphin and LDN-193189 inhibit BMP-mediated Smad, p38 and Akt signalling in C2C12 cells. Int. J. Biochem. Cell Biol. 42, 1802–1807. , , & (
- 2001) p21(ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells. J. Neurosci. Res. 63, 45–53. (
- 2003) Induction of neurite outgrowth in PC12 cells by the bacterial nucleoside N6-methyldeoxyadenosine is mediated through adenosine A2a receptors and via cAMP and MAPK signaling pathways. Biochem. Biophys. Res. Commun. 304, 795–800. , , , , & (
- 2008) Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitors. Bioorg. Med. Chem. Lett. 18, 4388–4392. , , , , , , , , & (
- 1992) The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J. Biol. Chem. 267, 5108–5113. & (
- 2009) Mevastatin-induced neurite outgrowth of neuroblastoma cells via activation of EGFR. J. Neurosci. Res. 87, 2138–2144. , & (
- 2000) Distinct signalling pathways mediate the cAMP response element (CRE)-dependent activation of the calcitonin gene-related peptide gene promoter by cAMP and nerve growth factor. Biochem. J. 345, 233–238. , & (
- 1994) Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J. Biol. Chem. 269, 6207–6214. , & (
- 2009) cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms. Biochem. Biophys. Res. Commun. 381, 247–252. , , , & (
- 1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl Acad. Sci. USA 73, 2424–2428. & (
- 2000) Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway. J. Biol. Chem. 275, 34433–34441. , , , , & (
- 2003) KCl potentiates forskolin-induced PC12 cell neurite outgrowth via protein kinase A and extracellular signal-regulated kinase signaling pathways. Neurosci. Lett. 347, 57–61. , & (
- 2008) Dorsomorphin, a selective small molecule inhibitor of BMP signaling, promotes cardiomyogenesis in embryonic stem cells. PLoS ONE 3, e2904. , , , , , , , & (
- 2010) In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors. ACS Chem. Biol. 5, 245–253. , , , , , , , & (
- 2001) BMP-2 augments FGF-induced differentiation of PC12 cells through upregulation of FGF receptor-1 expression. J. Cell Sci. 114, 1387–1395. , , & (
- 1997) TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465–471. , & (
- 1996) Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432–438. (
- 1996) Characterization of the bone morphogenetic protein-2 as a neurotrophic factor. Induction of neuronal differentiation of PC12 cells in the absence of mitogen-activated protein kinase activation. J. Biol. Chem. 271, 17360–17365. , , , & (
- 1999) Specific activation of the p38 mitogen-activated protein kinase signaling pathway and induction of neurite outgrowth in PC12 cells by bone morphogenetic protein-2. J. Biol. Chem. 274, 26503–26510. , , , , & (
- 2008) Cytosolic accumulation of gammaH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells. Exp. Mol. Med. 40, 276–285. , & (
- 2001) Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PC12 cells. J. Biol. Chem. 276, 18169–18177. , , & (
- 2010) Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev. 6, 270–281. , , , , , , , , & (
- 2000) BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. J. Biol. Chem. 275, 17647–17652. , , , & (
- 2006) The heat shock protein inhibitor KNK437 induces neurite outgrowth in PC12 cells. Neurosci. Lett. 410, 212–217. , , , , , & (
- 1998) The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase. Mol. Pharmacol. 54, 547–558. , & (
- 2010) Neurotrophic and cytoprotective action of luteolin in PC12 cells through ERK-dependent induction of Nrf2-driven HO-1 expression. J. Agric. Food. Chem. 58, 4477–4486. , , , & (
- 1998) Signal transduction. Taking the Rap. Nature 392, 553–554. (
- 2004) PKA phosphorylation of Src mediates Rap1 activation in NGF and cAMP signaling in PC12 cells. J. Cell Sci. 117, 6085–6094. , , & (
- 1995) Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J. Biol. Chem. 270, 13585–13588. , , & (
- 2008) Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening. Toxicol. Sci. 105, 106–118. , , & (
- 2008) Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth. Neurotoxicology 29, 361–376. & (
- 2001) Nerve growth factor stimulation of p42/p44 mitogen-activated protein kinase in PC12 cells: role of G(i/o), G protein-coupled receptor kinase 2, beta-arrestin I, and endocytic processing. Mol. Pharmacol. 60, 63–70. , & (
- 1987) Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J. Neurosci. 7, 3639–3653. & (
- 1996) Differential utilization of Trk autophosphorylation sites. J. Biol. Chem. 271, 20175–20181. , , , , , & (
- 2003) Dichlorodiphenyltrichloroethane suppresses neurite outgrowth and induces apoptosis in PC12 pheochromocytoma cells. Toxicol. Lett. 137, 175–183. & (
- 2002) Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 12, 258–266. & (
- 1991) Induction of neurite outgrowth of PC12 cells by an inhibitor of vacuolar H(+)-ATPase, bafilomycin A1. FEBS Lett. 294, 51–55. & (
- 1990) cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem. 59, 971–1005. , & (
- 2001) Induction of neurite outgrowth in PC12 cells by alpha-phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway. J. Biol. Chem. 276, 32779–32785. , & (
- 2002) Signaling pathways for PC12 cell differentiation: making the right connections. Science 296, 1648–1649. , , & (
- 1997) cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89, 73–82. , , , , & (
- 1994) Mechanism of activation of the TGF-beta receptor. Nature 370, 341–347. , , , & (
- 2001) Inhibition of BMP2-induced, TAK1 kinase-mediated neurite outgrowth by Smad6 and Smad7. Genes Cells 6, 1091–1099. , , , , , , , & (
- 2008) Effect of scoparone on neurite outgrowth in PC12 cells. Neurosci. Lett. 440, 14–18. , , , , & (
- 1998) The cyclic adenosine monophosphate-dependent protein kinase (PKA) is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor. J. Biol. Chem. 273, 8240–8247. , , , & (
- 2010) Induction of neurite outgrowth by alpha-phenyl-N-tert-butylnitrone through nitric oxide release and Ras-ERK pathway in PC12 cells. Free Radic. Res. 44, 645–654. , , , , , , , & (
- 2008a) BMP type I receptor inhibition reduces heterotopic [corrected] ossification. Nat. Med. 14, 1363–1369. , , , , , , , , , , , , , & (
- 2008b) Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4, 33–41. , , , , , , , & (
- 2008) Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins. Cell. Signal. 20, 1538–1544. , , , , & (
- 2001) Role of AMP-activated protein kinase in mechanism of metformin action. J. Clin. Invest. 108, 1167–1174. , , , , , , , , , , , , & (