c-Jun N-terminal kinase in synergistic neurite outgrowth in PC12 cells mediated through P90RSK
© Seow et al.; licensee BioMed Central Ltd. 2013
Received: 23 April 2013
Accepted: 5 December 2013
Published: 12 December 2013
Synergistic multi-ligand treatments that can induce neuronal differentiation offer valuable strategies to regulate and modulate neurite outgrowth. Whereas the signaling pathways mediating single ligand-induced neurite outgrowth, such as Akt, extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (P38), have been extensively studied, the mechanisms underlying multi-ligand synergistic neurite outgrowth are poorly understood. In an attempt to gain insight into synergistic neurite outgrowth, PC12 cells were treated with one of three combinations: pituitary adenylate cyclase-activating peptide (PACAP) with epidermal growth factor (EP), basic fibroblast growth factor (FP), or nerve growth factor (NP) and then challenged with the appropriate kinase inhibitors to assess the signaling pathways involved in the process.
Response surface analyses indicated that synergistic neurite outgrowth was regulated by distinct pathways in these systems. Synergistic increases in the phosphorylation of Erk and JNK, but not Akt or P38, were observed with the three growth factor-PACAP combinations. Unexpectedly, we identified a synergistic increase in JNK phosphorylation, which was involved in neurite outgrowth in the NP and FP, but not EP, systems. Inhibition of JNK using the SP600125 inhibitor reduced phosphorylation of 90 kDa ribosomal S6 kinase (P90RSK) in the NP and FP, but not EP, systems. This suggested the involvement of P90RSK in mediating the differential effects of JNK in synergistic neurite outgrowth.
Taken together, these findings reveal the involvement of distinct signaling pathways in regulating neurite outgrowth in response to different synergistic growth factor-PACAP treatments. Our findings demonstrate a hitherto unrecognized mechanism of JNK-P90RSK in mediating synergistic neurite outgrowth induced by the co-treatment of growth factors and PACAP.
KeywordsPC12 Synergistic NGF FGFb EGF PACAP Neurite outgrowth JNK P90RSK
The effects of synergistic activity have been gaining attention in the treatment of diseases such as cancer and AIDS [1, 2]. Drug or ligand synergy is defined as the joint action of two or more agents for which the result is greater than the sum of the actions of the individual parts. Synergistic therapeutic strategies therefore have the potential to accelerate the response to treatment, achieve higher efficacy, and potentially reduce the side effects associated with single treatment approaches [3–5]. Indeed, several studies have demonstrated the benefits of the co-administration of neurotrophic factors  and the combinatorial treatment of nerve growth factor (NGF) with glial cell-derived neurotrophic factor (GDNF)  or insulin-like growth factor (IGF)-1  in promoting synergistic axonal or neurite elongation. Axonal and dendritic outgrowth from the neuronal cell body is a key cellular aspect of neuronal differentiation that is critically important not just in development, but also in recovery from injuries and neurodegenerative diseases . Following nerve injury, the rate of nerve regeneration is critical, as a full functional recovery can be impeded by delayed regeneration . Thus, accelerating or facilitating neurite outgrowth through a synergistic approach may provide a valuable treatment strategy for patients with nerve injury. However, the mechanisms underlying such synergistic action are currently not well understood.
Rat pheochromocytoma PC12 cells have been widely used as a model to study neuronal differentiation. Upon treatment with ligands such as the NGF , basic-fibroblast growth factor (FGFb or FGF-2) , and pituitary adenylate cyclase-activating peptide (PACAP) , PC12 cells differentiate into sympathetic neuron-like cells characterized by long-term and stable neurite outgrowth. These ligands regulate neurite outgrowth through pathways such as the extracellular signal-regulated kinase 1/2 (Erk) [14, 15], p38 mitogen-activated protein kinase (P38) , c-Jun N-terminal kinase (JNK) [15, 17], and the phosphatidylinositol 3-kinase (PI3K) . In comparison, epidermal growth factor (EGF) promotes proliferation instead of differentiation in these cells [19, 20]. Studies have attributed this difference in cell fate to the kinetics of Erk activation, where transient or sustained Erk activation leads to proliferation or differentiation, respectively [14, 21]. Although EGF alone does not induce neurite outgrowth in PC12 cells, it has been found to synergize with cyclic adenosine monophosphate (cAMP)-elevating agents such as PACAP and forskolin, thereby enhancing neurite outgrowth [22, 23]. Consistent with the knowledge that Erk is important in regulating differentiation, enhanced Erk activity has also been observed in the synergy model . Similarly, cAMP-elevating agents have also been found to synergize with FGFb  and NGF [25, 26] to enhance neurite outgrowth, where both P38 and Erk have been found to regulate neurite outgrowth induced by NGF-cAMP [26, 27]. Whereas NGF, FGFb and EGF can all cooperate with cAMP-elevating agents to enhance neurite outgrowth, an interesting question is whether these three systems activate a common set of signaling pathways to mediate such synergy.
In this study, we investigated the activation and involvement of various signaling pathways in synergistic neurite outgrowth using three combinations of ligands: NGF-PACAP (NP), FGFb-PACAP (FP) and EGF-PACAP (EP). As expected, all three systems showed a synergistic phosphorylation of Erk concomitant with neurite outgrowth. Interestingly, JNK, but not Akt or P38, was also synergistically activated in all three systems. Unexpectedly, inhibition of JNK blocked neurite outgrowth in the NP and FP, but not EP, systems. This differential involvement of JNK was found to be dependent on the regulation of P90RSK activity. Thus, a JNK-P90RSK link was identified as a hitherto unrecognized mechanism mediating the synergistic effect in neurite outgrowth. Our results therefore demonstrate the involvement of distinct signaling pathways in regulating neurite outgrowth in response to different synergistic growth factor-PACAP stimulation.
Mouse recombinant NGF was purchased from Peprotech (Rocky Hill, NJ). Mouse recombinant EGF was purchased from Shenandoah Biotechnology (Warwick, PA). PACAP was purchased from American Peptide Company (Sunnyvale, CA). MEK inhibitor U0126, JNK inhibitor SP600125, PI3K inhibitor LY294002, and P38 inhibitor SB203580 were purchased from LC Laboratories (Woburn, MA). P90RSK inhibitor BRD7389 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies against phospho-specific Erk1/2 (Thr202/Tyr204) (pErk), pan-Erk1/2, phospho-specific JNK (Thr183/Tyr185) (pJNK), pan-JNK, phospho-specific P38 (Thr180/Tyr182) (pP38), phospho-specific Akt (Ser473) (pAkt), phospho-specific P90RSK (Ser380) (pP90RSK), and pan-RSK were purchased from Cell Signaling Technologies (Danver, MA). An antibody against phospho-specific c-Jun (Ser73) (pc-Jun) was purchased from Abnova (Taipei, Taiwan). Human recombinant FGFb and an antibody against actin were purchased from EMD Millipore (Billerica, MA). Horseradish peroxidase-conjugated secondary antibodies, Imperial Protein Stain and Hoechst were purchased from Thermo Scientific (Wilmington, DE).
Rat pheochromocytoma PC12 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO) and 5% Horse Serum (HS, Hyclone, Thermo Scientific). Cells were cultured with 100U/ml penicillin and 100 mg/ml streptomycin, and maintained in a humidified incubator with 5% CO2 at 37°C.
Western blot analyses
PC12 cells were seeded into the wells of 6-well plates pre-coated with poly-D-lysine at a density of 500,000 cells/well and cultured in growth medium for 48 hours. Following this, cells were incubated in serum-depleted medium (1% FBS, 0.5% HS) for an additional 16 hours. Cells were then simulated with individual or combinations of NGF, FGFb, EGF, and PACAP. For treatments with inhibitors, the cells were pre-incubated for 1 hour with the respective inhibitors prior to stimulations with the ligands. Cells were harvested within 1 hour after ligand stimulation. Treated cells were washed once with PBS and subsequently lysed in 2% sodium dodecyl sulfate (SDS). Protein concentrations in the total cell lysates were quantified using the microBCA assay (Pierce Biotechnology, Rockford, IL). The protein samples were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, blocked with 5% milk and probed with antibodies against phosphorylated (p)Erk (1:5000 dilution), pJNK (1:1000 dilution), pAkt (1:1000 dilution), pP38 (1:1000 dilution), pP90RSK (1:1000 dilution), pc-Jun (1:1000 dilution), total Erk (1:5000 dilution), total JNK (1:1000 dilution), total P90RSK (1:1000 dilution), and actin (1:10,000 dilution). Blots were stripped with Restore Western Stripping Buffer (Pierce Biotechnology) and re-probed for different proteins. The protein bands were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) on a ChemiDoc XRS system (Biorad, Hercules, CA). The band intensities were quantified using Quantity One 1-D Analysis software (Biorad). To enable comparisons of signals across different blots, lysates from NGF-PACAP–treated PC12 cells were used to generate a standard curve for each blot.
Measurement of neurite outgrowth
PC12 cells were seeded into the wells of 12-well plates at a density of 25,000 cells/well, and cultured as described for western blotting. After treatment with the respective ligands for 48 hours, the cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with ice-cold methanol for 15 minutes. The cell bodies were then stained with Imperial Protein Stain for 15 minutes and the nuclei with Hoechst stain for 5 minutes. The images of the cells were then captured using a Zeiss inverted fluorescent microscope (Zeiss Oberkochen, Germany). The length of the neurites was quantified using HCA-Vision software (CSIRO, North Ryde, NSW, Australia). The neurite quantification procedure, which involved neuron body detection, neurite detection, and neurite analysis, was performed as previously described . The neurite length obtained under control conditions (i.e., in the absence of NGF and PACAP) was subtracted from each treatment condition. Thereafter, the neurite length for each condition was normalized against that obtained for cells grown under 50 ng/ml of NGF, assigned an arbitrary value of 1.
Statistical significance was determined using the Student’s t-test and the respective results are displayed as the mean ± standard deviation (S.D.). All experiments and measurements were replicated at least three times.
Response surface analyses suggests that synergistic neurite outgrowth is regulated by discrete mechanisms in different systems
Synergistic phosphorylation of Erk & JNK upon combinatorial growth factor-PACAP treatment
We hypothesized that there was likely to be synergistic activation of the various kinases that regulate synergistic neurite outgrowth. To examine the pathways involved in regulating synergistic neurite outgrowth in these systems, we conducted a time-course to determine changes in the phosphorylation levels of four kinases—Akt, Erk, JNK, and P38—upon NGF, PACAP, and NP treatments. The kinases were activated throughout the entire 1 hour time-course (Additional file 1: Figure S1). Thus, for convenience, subsequent analyses were performed only at 20 and 60 minutes time-points.
Similarly, sustained activation of JNK by NGF was observed (Figure 2c). In addition, we made the novel discovery that JNK was also synergistically phosphorylated upon combinatorial NP treatment (Figure 2c, & Additional file 3: Figure S3b) and it was sustained for up to 1 hour post-stimulation. On the contrary, using the same analyses, synergistic phosphorylation of P38 (Additional file 2: Figure S2a, & S2b) and Akt (Additional file 2: Figure S2a, & S2c) were not observed in the NP system.
The total protein levels of Erk, JNK, P38 and Akt upon treatment with single ligand or combinations of the growth factors and PACAP were unchanged across all conditions and time-points (Additional file 5: Figure S5).
Erk is required for neurite outgrowth in all 3 systems whereas JNK is required only for the NP and FP, but not EP, systems
As expected, inhibition of the non-synergistically activated nodes, P38 and Akt, by SB203580, and LY294002, respectively, did not block neurite outgrowth in all three systems (Figure 4a(iii)-(iv), b, c, & Additional file 6: Figure S6). Likewise, cells treated with doses of the inhibitors at concentrations higher than 20 μM resulted in high levels of cytotoxicity (data not shown). The positive controls for SB203580 and LY294002 are shown in Additional file 7: Figure S7c and S7d, respectively.
Critically, these results also suggest that these systems utilize distinct pathways to regulate neurite outgrowth and that not all synergistically phosphorylated kinases are relevant to neurite outgrowth.
P90RSK is a downstream target of both Erk & JNK in the NP & FP systems but is only downstream of Erk in the EP system
Having found that JNK was involved in neurite outgrowth in the NP and FP, but not EP, systems, we sought to identify the downstream targets that may be involved in mediating this differential requirement of JNK. Among the many downstream effectors of JNK, P90RSK has been recently shown to be involved in neurite outgrowth and PC12 cells differentiation [32–34]. Thus, we examined if P90RSK was synergistically phosphorylated and if it was involved in JNK-mediated neurite outgrowth.
In addition to JNK, P90RSK has also been reported to be a downstream target of Erk [37, 38]. Unlike the case for JNK inhibition, inhibition of Erk activation with U0126 suppressed P90RSK phosphorylation in all three systems (Figure 7), providing further support for the role of P90RSK as an important mediator of neurite outgrowth. The total levels of Erk, JNK, and P90RSK were unchanged during the combinatorial growth factor-PACAP treatments both in the presence and absence of the inhibitors (Additional file 9: Figure S9).
cAMP-elevating agents have long been known to synergize with NGF [25, 26], FGFb , and EGF [22, 23] to enhance neurite outgrowth. Although the pathways used by these individual ligands to regulate neurite outgrowth have been widely studied, little is known about the mechanisms underlying synergistic neurite outgrowth. RSM-based analyses provide a means to quantitatively compare the degree of synergism between different treatments . By such analyses, the degree of synergism in the EP system was found to be higher than those in the NP and FP systems, suggesting that different signaling pathways may regulate neurite outgrowth in these systems.
To determine the pathways involved in synergistic neurite outgrowth, four kinases were examined, each widely reported to be involved in PC12 cells differentiation: Erk [14, 39, 40], P38 [16, 39], JNK [17, 41], and Akt [18, 40, 42]. Interestingly, our results showed that Akt and P38 were activated following ligand stimulation but not involved in neurite outgrowth in all three systems. In agreement with this, inhibition of these two kinases also failed to suppress NGF-induced PC12 cells neurite outgrowth. These results were consistent with some of the earlier reports exploring neurite outgrowth [43–45] but not others [15–18, 39–42]. A recent systems-based study revealed a two-dimensional Erk-Akt signaling code that was critical in governing PC12 cells proliferation and differentiation . Thus, the controversy surrounding the involvement of P38 and Akt would be more adequately addressed using systems-based approaches in the future.
The sustained activation of Erk has been widely reported to be required for neurite outgrowth during differentiation [14, 21, 27, 31]. Consistent with these reports, synergistic and sustained Erk phosphorylation was found to be involved in neurite outgrowth in all three growth factor-PACAP systems. This was especially evident in the EP system, where transient Erk activation was observed following treatment with EGF or PACAP alone. Similarly, synergistic and sustained JNK phosphorylation was observed in all three systems. Remarkably, inhibition of JNK led to reduced neurite outgrowth in the NP and FP systems, but enhanced outgrowth in the EP system. Although a previous study has found sustained JNK activation to be sufficient to induce PC12 cells differentiation , our results showed that sustained JNK activation in the EP system is insufficient to induce neurite outgrowth. These seemingly contradictory findings could imply that the kinetics of JNK activation alone is insufficient to determine if cells undergo differentiation. It is likely that JNK acts in conjunction with other signaling nodes to form a signaling network that regulates neurite outgrowth. Nonetheless, to the best of our knowledge, this is the first report demonstrating the involvement of JNK phosphorylation in synergistic neurite outgrowth.
We have shown that both Erk and JNK were synergistically phosphorylated in all three systems. This may occur through shared common upstream effectors  or through independent upstream effectors, such as PKA and Epac [49–51]. In preliminary experiments, we observed the involvement of PKA in neurite outgrowth in the EP but not NP system (data not shown); however, a complete understanding of the contribution of PKA and Epac in Erk and JNK activation remains to be determined.
Although synergistic JNK phosphorylation was observed in all three systems, it was not found to be involved in synergistic neurite outgrowth in the EP system. This suggests a possible difference in downstream signaling. P90RSK, which had previously been found to be required for PC12 cells differentiation , was also found to be synergistically activated in all three systems in our study. Interestingly, P90RSK was activated by JNK in the NP and FP, but not EP, systems. Although JNK-mediated activation of P90RSK has not been widely reported, it has been observed following ultraviolet exposure , insulin treatment , or transforming growth factor alpha treatment . Consistent with previous findings , P90RSK was also regulated by Erk in our study. The co-regulation of targets by Erk and JNK is not uncommon, with previous studies showing that these two kinases regulate many common targets, including transcription factors [15, 54, 55], immediate early genes  and differentiation-specific genes [15, 56, 57]. Despite this, results from several studies have suggested that the binding sites of P90RSK for Erk and JNK are likely to be different [34, 58], further indicating that P90RSK may be discretely regulated by the two kinases. Our finding of the differential regulation of P90RSK in the NP and EP systems in this study strongly suggests that these synergistic systems can serve as excellent models to decipher the mechanistic regulation of P90RSK by its upstream kinases, Erk and JNK. The contributions of Erk, JNK and P90RSK in the mechanism of axonal outgrowths of neurons in vivo and in vitro will require further clarification in future studies.
In conclusion, our study has demonstrated distinct pathways involved in synergistic neurite outgrowth in different systems. Importantly, our findings of the underlying pathways involved in these systems have two key implications. First, some kinases such as JNK may be synergistically activated by multiple ligands but yet not necessarily involved in the synergistic neurite outgrowth process and that its involvement in neurite outgrowth is dependent on its interaction with P90RSK. Second, in the EP system, the increased synergy in neurite outgrowth and lack of JNK requirement for regulating the process suggest that PACAP synergizes differently with different growth factors to enhance neurite outgrowth. These findings reveal that synergistic of neurite outgrowths induced by multiple ligands involves the interplay of a network of signals.
Nerve growth factor
Basic-fibroblast growth factor
Epidermal growth factor
Pituitary adenylate cyclase-activating peptide
Response surface model
Extracellular signal-regulated kinase 1/2
c-Jun N-terminal kinase
P38 mitogen-activated protein kinases
Cyclic adenosine monophosphate
Protein kinase A
Exchange protein activated by cAMP.
The authors would also like to acknowledge the Singapore–Massachusetts Institute of Technology Alliance for providing the necessary funding, and the Department of Biochemistry for the laboratory facilities to carry out the study.
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