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Research ArticleArticle

Neuritogenic Activity of Tetradecyl 2,3-Dihydroxybenzoate Is Mediated through the Insulin-Like Growth Factor 1 Receptor/Phosphatidylinositol 3 Kinase/Mitogen-Activated Protein Kinase Signaling Pathway

Ruiqi Tang, Lijuan Gao, Makoto Kawatani, Jianzhong Chen, Xueli Cao, Hiroyuki Osada, Lan Xiang and Jianhua Qi
Molecular Pharmacology August 2015, 88 (2) 326-334; DOI: https://doi.org/10.1124/mol.115.097758

Abstract

Tetradecyl 2,3-dihydroxybenzoate (ABG-001) is a lead compound derived from neuritogenic gentisides. In the present study, we investigated the mechanism by which ABG-001 induces neurite outgrowth in a rat adrenal pheochromocytoma cell line (PC12). Inhibitors of insulin-like growth factor 1 (IGF-1) receptor, phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK) 1/2 significantly decreased ABG-001–induced neurite outgrowth. Western blot analysis revealed that ABG-001 significantly induced phosphorylation of IGF-1 receptor, protein kinase B (Akt), ERK, and cAMP responsive element-binding protein (CREB). These effects were markedly reduced by addition of the corresponding inhibitors. We also found that ABG-001–induced neurite outgrowth was reduced by protein kinase C inhibitor as well as small-interfering RNA against the IGF-1 receptor. Furthermore, like ABG-001, IGF-1 also induced neurite outgrowth of PC12 cells, and low-dose nerve growth factor augmented the observed effects of ABG-001 on neurite outgrowth. These results suggest that ABG-001 targets the IGF-1 receptor and activates PI3K, mitogen-activated protein kinase, and their downstream signaling cascades to induce neurite outgrowth.

Introduction

Neurotrophic factors such as nerve growth factors (NGFs) participate in neuronal development and in the survival and functional maintenance of neurons (McAllister 2001). Several studies have indicated that reduced neurotrophic support is a contributing factor in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Dawbarn and Allen, 2003). Therefore, neurotrophic factors are strong-candidate therapeutic agents for chronic neurodegenerative diseases. Unfortunately, NGF cannot penetrate the blood-brain barrier because of its size, and this limitation hinders its applications in clinical therapy. As such, screening for NGF-mimicking chemical compounds with low molecular weights and the ability to pass through the blood-brain barrier is an active area of research.

Natural compounds such as termitomycesphins A–D (Qi et al., 2000), linckosides A–E (Qi et al., 2002, 2004), panax ginseng (Yamazaki et al., 2001), and nardosinone (Li et al., 2003) as well as synthetic compounds such as 4,5-bis-epi-neovibsanin A (Chen et al., 2010), xaliproden (Sabbagh, 2009), and synthetic verbena chalcone derivatives (Clement et al., 2009) have recently been shown to have NGF-mimicking or NGF-enhancing activity related to neurite outgrowth in a rat adrenal pheochromocytoma cell line (PC12). Previous studies have identified diverse molecular signaling pathways participating in the neuron-protective activities of small natural compounds (Macready et al., 2009; Weng et al., 2011). These pathways include selective actions of a number of protein and lipid kinase signaling cascades, the most now of which involve the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) pathways that regulate prosurvival transcription factors and gene expression.

Gentiana rigescens Franch (G. rigescens) is a traditional Chinese medicine that is believed to remove heat and dampness from the human body. It is used to treat hypertension, cholecystitis, acute infective hepatitis, and cystitis. Pharmacodynamic studies have indicated that G. rigescens has anti-inflammation, antipathogen, antioxidant, antitumor, and proimmunity functions (Tang et al., 1977). In our previous study, we described the isolation of gentisides A−K, which are new neuritogenic compounds, from G. rigescens (Gao et al., 2010a,b). These gentisides showed good neuritogenic activities in PC12 cells. To study their structure-activity relationships and to facilitate the discovery of the lead compounds in gentisides, we synthesized more than a hundred derivatives of gentisides and tested their neuritogenic activities in PC12 cells. We found that ABG-001 (tetradecyl 2,3-dihydroxybenzoate; Fig. 1A) exhibited strong NGF-mimicking effects via the extracellular signal-regulated kinase (ERK) signaling pathway (Luo et al., 2011). However, the molecular target of gentisides and the mechanism behind induction of neurite outgrowth in PC12 cells remain unknown.

Fig. 1.
Fig. 1.

(A) Chemical structure of ABG-001. (B) Neurite outgrowth in PC12 cells of ABG-001 at different concentrations. (C) Percentage of viable PC12 cells after treatment with ABG-001 for 48 hours. (D) Effects of TrkA inhibitor on the neurogenic effects of ABG-001 in PC12 cells. Percentage of neurite outgrowth as monitored using a phase-contrast microscope 48 hours after ABG-001 treatment. Neurites were identified as cells bearing neurites that were at least twice the cell diameter. (Control: DMSO, 0.5%; positive control: NGF, 40 ng/ml). *Significantly different from the control group at the same time point at P < 0.05. ***Significantly different from the control group at the same time point at P < 0.001. ###Significantly different from the NGF group at the same time point at P < 0.001.

In the present study, we used inhibitors of the NGF signaling pathway and small-interfering RNAs (siRNAs) in combination with Western blot assays to investigate the mechanism behind improvements in neurite outgrowth induced by ABG-001. We report that ABG-001 may target the insulin-like growth factor 1 (IGF-1) receptor and activate the PI3K, protein kinase C (PKC)/MAPK, and the associated downstream signaling cascades to induce neuritogenic activity.

Materials and Methods

Chemicals and Reagents.

ABG-001 was synthesized and purified using high-pressure liquid chromatography in our laboratory (Luo et al., 2011). Dimethylsulfoxide (DMSO); NGF; the PI3K inhibitors LY294002 (2-morpholin-4-yl-8-phenylchromen-4-one) and wortmannin; the mitogen-activated protein-extracellular signal-regulated kinase (MEK)/ERK inhibitors U0126 [(2Z,3Z)-2,3-bis[amino-(2-aminophenyl)sulfanylmethylidene]butanedinitrile] and PD98059 [2-(2-amino-3-methoxyphenyl)chromen-4-one]; the IGF-1 receptor kinase inhibitor T9576 (picropodophyllotoxin); the PKC inhibitors GF109203X (bisindolylmaleimide I), Gö6983 [3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione]); and the protein kinase A (PKA) inhibitor H-89 ([N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide]) were purchased from Sigma-Aldrich (St. Louis, MO). Ras inhibitors (sulindac sulfide and farnesylthiosalicylic acid) were purchased from Sigma-Aldrich and Cayman Chemical (Ann Arbor, MI). The phospholipase C (PLC) inhibitors U73122 (1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione) and U73343 (1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), the PKC inhibitors Ro318220 [3-(1-(3-(amidinothio) propyl-1H-indol-3-yl))-3-(1-methyl-1H-indol-3-yl)maleimide], the Jun N-terminal kinase (JNK) inhibitor SP600125 (1,9-pyrazoloanthrone), the p38 MAPK inhibitor SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine), the tyrosine kinase A (TrkA) inhibitor K252a [(9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3ʹ,2ʹ,1ʹ-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one], the insulin receptor inhibitor HNMPA(AM)3 [hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester], the IGF-1 inhibitor AG1024 [2-(3-bromo-5-(tert-butyl)-4-hydroxybenylidene)malononitrile], and the Raf inhibitor AZ628 [3-(2-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxoquinazolin-6-yl)amino]phenyl]benzamide] were purchased from Santa Cruz Biotechnology (Dallas, TX). IGF-1 was purchased from Sino Biologic Company (Beijing, People’s Republic of China).

Cell Culture.

The rat adrenal pheochromocytoma cell line PC12 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, People’s Republic of China). PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Scientific, Waltham, MA) containing 4 mM glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, and 1 mM sodium pyruvate and supplemented with 10% horse serum and 5% fetal bovine serum (Invitrogen, Grand Island, NY) in a 5% CO2 incubator at 37°C. Before treatment, cells were subcultured and allowed to attach overnight.

Neurite Outgrowth Assay.

Briefly, 2 × 104 PC12 cells were seeded in the wells of a 24-well microplate and cultured under a humidified atmosphere of 5% CO2 at 37°C. The medium was replaced with 1 ml of serum-free DMEM containing the test sample or DMSO (0.5%) after 24 hours. NGF was used as a positive control. The number of neurite-bearing cells was measured by counting cells in three arbitrary areas on the 24-well plate containing at least 100 single cells (not aggregated). A cell was identified as positive for neurite outgrowth if the outgrowths were at least twice the cell diameter (Jeon et al., 2010a,b). Cells were visualized using phase contrast (200-fold magnification) with an Olympus microscope (Model CK-2; Olympus China, Beijing, People’s Republic of China).

About 100 cells were counted from a random site, and each experiment was repeated three times. If the neurite outgrowth of 100 cells observed in an experiment were twice the cell diameter, then the outgrowth was considered to be 100%. The results are expressed as mean ± S.E.M. For experiments involving inhibitors, we first performed a dose-dependent investigation. The optimum concentration of inhibitors was then used to conduct the subsequent experiments.

Analysis of Cell Viability by MTT Assay.

Cell viability was determined based on mitochondria-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to purple formazan. Briefly, the cells were incubated with ABG-001 at concentrations of 0, 0.3, 0.6, or 1 μM for 48 hours. The medium was carefully removed by aspiration, and 0.5 ml of fresh medium containing MTT (0.2 mg/ml) was added to each well, and the plates were incubated at 37°C for 2 hours. The medium was then completely removed, and 0.2 ml of DMSO was added to each well to solubilize the formazan crystals. The resultant formazan was detected at 570 nm using a plate reader. All experiments were repeated at least three times.

Real-Time Polymerase Chain Reaction Analysis.

Cells treated with siRNA against the IGF-1 receptor and ABG-001, respectively, were collected. The total RNA was extracted using TRIzol reagent (Beijing Cowin Biotech; Beijing, People’s Republic of China), and the RNA content was determined using a spectrophotometer. Transcription was performed using 2.5 μg of total RNA, Oligo(dT)20 primers, and reverse transcriptase (Beijing Cowin Biotech). Transcript levels were quantified by real-time polymerase chain reaction (AB SCIEX, Framingham, MA) and SYBR Premix EX Taq (Takara, Otsu, Japan). The polymerase chain reaction primers for rat IGF-1 receptor and 18S RNA were as follows: for IGF-1 receptor, sense: 5′-ATG GCT TCG TTA TCC ACG AC-3′, and anti-sense: 5′-CGA ATC GAT GGT TTT CGT TT-3′; for 18S RNA, sense: 5′-TAA CCC GTT GAA CCC CAT T-3′, and anti-sense: 5′-CCA TCC AAT CGG TAG TAG CG-3′. The cDNA was then amplified using Takara SYBR Premix Ex Taq under the following conditions: 95°C for 2 minutes, followed by 40 cycles for 15 seconds at 95°C, 15 seconds at 54.2°C, and 20 seconds at 68°C. All results were normalized to 18S RNA levels, and the relative mRNA transcript levels were calculated using the ΔΔCt formula. All samples were run in triplicate, and the average values were calculated.

Western Blot Analysis.

Approximately 1 × 106 PC12 cells were seeded in a 60-mm culture dish containing 5 ml DMEM and incubated for 24 hours. To investigate the dose-dependent effect of ABG-001, various ABG-001 concentrations were added to the cultures at concentrations of 0, 0.3, 0.6, or 1.0 μM, after which they were incubated for 30 minutes or 16 hours. To investigate the time-dependent effects of ABG-001, ABG-001 was added at a final concentration of 1 μM, after which the cultures were incubated for specific time periods.

To investigate the effects of siRNA IGF-1 receptor on neurite outgrowth induced by ABG-001, ABG-001 was added after cell transfection of the negative control siRNA against IGF-1 receptor for 6 hours.

To prepare protein lysates, cells were collected in lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, and 1% phosphatase inhibitor) and then sonicated using an ultrasonicator (Ningbo Scientz Biotechnology, Ningbo, People’s Republic of China). The supernatant containing the proteins was collected by centrifugation at 12 × 103 rpm for 15 minutes. The protein concentration was measured using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA) at 595 nm. Protein lysates (15 μg) were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. The membrane was incubated with primary antibodies followed by horseradish peroxidase–conjugated secondary antibodies. Antigens were visualized using chemiluminescent substrates (Amersham Biosciences/GE Healthcare, Little Chalfont, United Kingdom).

The primary antibodies used for immunoblotting are as follows: anti–44/42 MAPK antibody, anti–phospho-p44/42 MAPK (Thr202/Tyr204) polyclonal antibody, anti–IGF-1 receptor β antibody, anti–phospho-IGF-1 receptor β (Tyr1135/1136)/insulin receptor β (Tyr1150/1151), anti–phospho-CREB (Ser133), anti-CREB and anti-Akt antibodies (Cell Signaling Technology, Beverly, MA), anti–phospho-Akt (Ser473) (Abcam, Hong Kong, People’s Republic of China), and GAPDH antibody (Beijing Cowin Biotech). The secondary antibodies used in this study are as follows: horseradish peroxidase–linked anti-rabbit and anti-mouse IgGs (Beijing Cowin Biotech).

Cell Transfection.

PC12 cells were transfected with 5-carboxy-fluorescein (FAM)–labeled siRNA to investigate transfection efficiency. A concentration of 120 nM, at which the transfection efficiency reached 90%, was used to perform the experiment. The following primer sequences were used to generate siRNAs targeting the rat IGF-1 receptor and the negative control (Shanghai GenePharma Company, Shanghai, People’s Republic of China): for IGF-1 receptor-530, sense: 5′-GCG GUG UCC AAU AAC UAC ATT-3′, anti-sense: 5′-UGU AGU UAU UGG ACA CCG CTT-3′; for negative control, sense: 5′-UUC GAA CGU GUC ACG UTT-3′, anti-sense: 5′-ACG UGA CAC GUU CGG AGA ATT-3′.

Transfection of PC12 cells with siRNA was performed according to the manufacturer’s protocol (Invitrogen). Briefly, the day before transfection, 2 × 104 cells were seeded in each well of 24-well plates and allowed to reach 70–90% confluence in growth medium without antibiotics. Then siRNA against IGF-1 receptor or the negative control siRNA was used at a concentration of 120 nM with Lipofectamine 2000 (Invitrogen) as the transfection agent. After 6 hours of transfection, the medium in the plates was replaced with fresh medium containing 1 μM ABG-001 and incubated for an additional 24 hours. Cell morphologic features were observed and recorded using a microscope fitted with a camera. The lengths of neurites were measured using ImageJ software (National Institutes of Health, Bethesda, MD).

Statistical Analysis.

All experiments were independently performed three times, and each experiment was conducted with triplicate samples. Data are presented as mean ± S.E.M. The statistically significant differences between groups were determined by analysis of variance, followed by two-tailed multiple t tests with Student-Newman-Keuls through SPSS, biostatistics software (IBM, Chicago, IL). P < 0.05 was considered statistically significant.

Results

ABG-001 Induces Neurite Outgrowth in PC12 Cells.

Figure 1B shows the neuritogenic activity of ABG-001 in PC12 cells. ABG-001 induced neurite outgrowth in PC12 cells in a dose-dependent manner. The percentages of neurite-bearing cells treated with 0.3, 0.6, and 1.0 μM ABG-001 for 48 hours were 57.7 ± 5.4%, 70.1 ± 3.6%, and 83.9 ± 5.8%, respectively, and were significantly higher than that of the control group (P < 0.001). This result suggests that ABG-001 has significant effects on neurite outgrowth in PC12 cells and is consistent with the findings of a previous study (Luo et al., 2011).

Effects of ABG-001 on PC12 Cell Viability.

The effect of ABG-001 on PC12 cell viability was determined by MTT assay. Figure 1C illustrates that PC12 cells treated with ABG-001 concentrations of 0.3, 0.6, and 1 μM for 48 hours show viabilities of 82.6 ± 4.7%, 78.5 ± 11.3%, and 75.5 ± 7.1%, respectively. These results indicate that ABG-001 is weakly cytotoxic toward PC12 cells.

Effects of ABG-001 on the TrkA Signaling Pathway.

We investigated the function of the TrkA receptor in neurite outgrowth induced by ABG-001 using the TrkA receptor inhibitor K252a. Application of this inhibitor did not affect the neuritogenic activity of ABG-001 in PC12 cells (Fig. 1D; Supplementary Fig. 1A).

Effects of ABG-001 on the MAPK/ERK/CREB Signaling Pathway.

We investigated the function of ERK1/2 activation in ABG-001–induced neurite outgrowth in PC12 cells. U0126 significantly attenuated the percentage of neurite-bearing cells from an initial value of 71.8 ± 5.5% to 32.4 ± 2% after the cells had been treated with 1 μM ABG-001 (Fig. 2, A and B; Supplemental Fig. 2B). The average of neurite length of PC12 cells induced by NGF and ABG-001 was significantly reduced by U0126 (Fig. 2C).

Fig. 2.
Fig. 2.

(A) Photomicrographs of PC12 cells after treatment with ABG-001 and U0126 for 48 hours: (a) Control (0.5% DMSO), (b) NGF (40 ng/ml), (c) 1 μM ABG-001, (d) 40 ng/ml NGF + 10 μM U0126, and (e) 1 μM ABG-001 + 10 μM U0126. (B) Effect of U0126 on the neurite outgrowth in PC12 cells induced by ABG-001 at 1 μM. (C) The average of neurite length for control group was 0.175 ± 0.025 (μm); NGF at 40 ng/ml, 1.303 ± 0.117 (***); ABG-001 at 1 μM, 1.587 ± 0.158 (***); NGF + U0126, 0.717 ± 0.054 (###); ABG-001 + U0126, 0.178 ± 0.031($$$). ABG-001 induced phosphorylation of ERK1/2 in a (D) dose- and (E) time-dependent manner (the cells treated with each agent for 16 hours in dose-dependent experiment). (F) ABG-001–stimulated CREB phosphorylation. NGF was used as a positive control, and ERK and GAPDH antibodies were used as loading controls. ***Significantly different from control group at the same time point at P < 0.001. ###,$$$Significantly different from NGF or ABG-001–treated group at P < 0.001.

Furthermore, we determined the effects of ABG-001 on ERK phosphorylation at the protein level. ERK1/2 phosphorylation was enhanced by ABG-001 at concentrations of 0, 0.6, 1.0, and 1.2 μM and reduced by U0126 (Fig. 2D). ERK1/2 phosphorylation induced by ABG-001 began 8 hours after treatment, and then gradually decreased after 48 hours (Fig. 2E).

CREB phosphorylation induced by ABG-001 was initiated 8 hours after treatment and was maintained for 48 hours (Fig. 2F). Parallel blots were run and probed with antibodies to detect total ERK1/2, CREB, and GAPDH levels. The results demonstrated that all proteins had been loaded at equivalent levels. These findings indicate that ERK/CREB signaling is involved in ABG-001–induced neurite outgrowth of PC12 cells.

Effects of ABG-001 on the PI3K/Akt Signaling Pathway.

PI3K is critical for NGF-dependent survival of sympathetic neurons, and Akt, a major effecter, has the important role of integrating various survival signaling cascades (Crowder and Freeman, 1998; Pierchala et al., 2004). Here, specific inhibitors of PI3K, namely, LY294002 and wortmannin, were used to examine the effects of ABG-001 on the PI3/Akt signaling pathway.

ABG-001–induced neurite outgrowth and the average length of PC12 cell neuritis was markedly reduced by 20 μM LY294002 (Fig. 3, A and B). LY294002 attenuated the percentage of neurite-bearing cells from an initial value of 70.9 ± 3.3% to 42.75 ± 1.2% after the cells had been treated with 1 μM ABG-001 (Fig. 3C; Supplemental Fig. 3A).

Fig. 3.
Fig. 3.

(A) Effect of ABG-001 on the PI3K/Akt signaling pathway in PC12 cells. Morphologic changes in PC12 cells after treatment with ABG-001 and LY294002 for 48 hours: (a) Control (0.5% DMSO), (b) positive control (NGF 40 ng/ml), (c) 1 μM ABG-001, (d) 40 ng/ml NGF + 20 μM LY294002, and (e) 1 μM ABG-001 + 20 μM LY294002. (B) The average neurite length for the control group was 0.190 ± 0.042 (μm); NGF at 40 ng/ml, 1.576 ± 0.276***; ABG-001 at 1 μM, 1.486 ± 0.231***; NGF + LY294002, 0.717 ± 0.054###; ABG-001 + LY294002, 0.178 ± 0.031$$$. (C) Effects of LY294002 on ABG-001–induced neurite outgrowth. (D and E) ABG-001–stimulated phosphorylation of Akt in a time- and dose-dependent manner (the cells treated with each agent for 30 minutes in dose-dependent experiment). NGF was used as a positive control, and Akt and GAPDH antibodies were used as loading controls. ***Significant difference compared with control group at P < 0.001. ###,$$$Significant difference compared with NGF or ABG-001–treated group at P < 0.001.

Next, we investigated Akt phosphorylation at the protein level at multiple time points (Fig. 3D). Akt phosphorylation level increased with increase in treatment time in dose-dependent manner at ABG-001 concentrations of 0.0, 0.6, 1.0, and 1.2 μM. Furthermore, Akt phosphorylation was significantly decreased by LY294002 treatment (Fig. 3E). ABG-001–induced neurite outgrowth was also reduced by wortmannin (Supplemental Fig. 3B). The inhibitory effects of LY294002 were stronger than those of wortmannin. These results suggest that PI3K/Akt may exert an important effect on ABG-001–induced neuronal differentiation of PC12 cells.

Effects of ABG-001 on the Insulin/IGF-1 Signaling Pathway and PC12 Cell Differentiation.

To determine the target of ABG-001, we focused on receptors located upstream of PI3K. As expected, HNMPA(AM)3, the specific inhibitor of insulin receptor, slightly attenuated the percentage of neurite-bearing cells from an initial value of 69.4 ± 4.0% to 59.7 ± 4.3% after treatment with 1 μM ABG-001 (Fig. 4A; Supplemental Fig. 4A). However, the percentage of neurite-bearing cells was reduced from an initial value of 60.9 ± 5.5% to 41.9 ± 3.7% after treatment with the IGF-1 inhibitor AG1024 (Fig. 4B; Supplemental Fig. 4B).

Fig. 4.
Fig. 4.

(A, B, and C) Effect of insulin/IGF-1 inhibitors on the neurogenic effects of ABG-001 in PC12 cells. Cells were pretreated with 10 μM HNMPA(AM)3, 0.1 μM AG1024, 0.1μM T9576, specific inhibitors of insulin, and IGF-1 inhibitors for 30 minutes, followed by addition of 1 μM ABG-001. (D) Time- and (E) dose-dependent changes in phosphorylation of the IGF-1 receptor (the samples were obtained after treatment with ABG-001 for 5 minutes). NGF was used as a positive control, and IGF-1 receptor and GAPDH antibodies were used as loading controls. (F) Effects of IGF-1 and T9576 on the neurite outgrowth of PC12 cells. ***Significantly different from the NGF-treated group at the same time point at P < 0.001. ###Significantly different from the ABG or IGF-1–treated groups at the same time point at P < 0.05 or P < 0.01.

Changes in IGF-1 receptor phosphorylation were further observed after treatment with ABG-001. As shown in Fig. 4, D and E, ABG-001 induced dose-dependent enhancements in IGF-1 receptor phosphorylation, which peaked at 10 minutes; these enhancements were significantly reduced by AG1024. Furthermore, application of the tyrosine kinase IGF-1 receptor inhibitor T9576 led to a significant decrease in the percentage of neurite outgrowth from PC12 cells from 83 ± 2.3% to 42.6 ± 2.3% (Fig. 4C; Supplemental Fig. 4C).

To determine whether the IGF-1 signaling pathway is the primary mechanism by which ABG-001 affects PC12 cells, we examined the effects of IGF-1 on neurite outgrowth of these cells. As expected, IGF-1 significantly enhanced the neurite outgrowth of PC12 cells, just as ABG-001 and NGF did. This neurotrophic effect of IGF-1 on PC12 cells could also be inhibited by the IGF-R inhibitor T9576 (Fig. 4F). These results suggest that ABG-001, like IGF-1, targets the IGF-1 receptor and regulates the expression of downstream genes to induce NGF-like effects in PC12 cells.

Effects of Knockdown IGF-1 Receptor by siRNA on Neurite Outgrowth after Treatment with ABG-001.

To further verify that ABG-001 targets the IGF-1 receptor, we first used FAM-siRNA to determine the best siRNA transfection concentration. Photomicrographs of PC12 cells after transfection with FAM-siRNA are displayed in Fig. 5, A and B. Approximately 90% of the PC12 cells produced fluorescence at 120 nM. Therefore, we used this dose to transfect IGF-1 receptor siRNA into PC12 cells for 6 hours and then treated with 1 μM ABG-001.

Fig. 5.
Fig. 5.

Effect of IGF-1 receptor siRNA on the neurogenic effects of ABG-001 in PC12 cells. Microphotograph of PC12 cells after transfection with (A and B) FAM-siRNA and (C and D) IGF-1 receptor siRNA. (E) Change of neurite length in PC12 cells after treatment with IGF-1 receptor siRNA. (F) Gene expression of IGF-1 receptor after treatment with IGF-1 receptor siRNA and ABG-001. (G) Neurite outgrowth percentage of PC12 cells after treatment with IGF-1 receptor siRNA and ABG-001. (H) Western blot analysis for IGF-1 receptor after treatment with siRNA of IGF-1 receptor. Cells were transfected with lipofectamine 2000 and 120 nM IGF-1 receptor siRNA for 6 hours and then treated with 1 μM ABG-001. The ABG-001 treatment group was used as a positive control. **,***Significantly different from the ABG-001–treated group at the same time point at P < 0.01, P < 0.001. #Significantly different from negative siRNA-treated group at the same time point at P < 0.05.

The lengths of neurites induced by ABG-001 were significantly reduced by IGF-1 receptor siRNA from 48.66 ± 4.17 to 28.22 ± 1.37 μm (Fig. 5, C–E). The percentages of neurite outgrowth and the gene expression level of IGF-1 receptor were also significantly decreased after treatment with IGF-1R siRNA (Fig. 5, F and G). Furthermore, the total protein level of IGF-1 receptor and phosphorylation of IGF-1 receptor were both significantly reduced by treatment with siRNA against IGF-1 receptor (Fig. 5H). At the same time, phosphorylation of Akt and ERK were significantly lowered by siRNA against IFG-1 receptor (Fig. 6F). These results are consistent with our earlier observations that ABG-001 acts through the IGF-1 receptor.

Fig. 6.
Fig. 6.

(A and B) Effect of PKC inhibitors on the neurogenic effects of ABG-001 in PC12 cells. (C and D) Effect of NGF on the neurogenic effects of ABG-001 in PC12 cells treated for 24 hours. (E) Proposed mechanism of ABG-001 in induction of neurite outgrowth in PC12 cells. ABG-001 induces neurite outgrowth through activation of the IGF-1 receptor, PI3K/Akt-PKC, and MAPK/ERK dependent pathways in PC12 cells (Figs. 2–6). (F) The change in Akt and ERK phosphorylation after treatment with siRNA against IGF-1 receptor. Cells were pretreated with 0.1 μM Ro318220 and 5 μM Gö6983, specific inhibitors of PKC, for 30 minutes and then administered 0.3 or 1 μM ABG-001. NGF was used as a positive control. **Significantly different from the control group at the same time point at P < 0.01. #,##,$$Significantly different from the NGF or ABG-001–treated group at the same time point at P < 0.05 or P < 0.01.

Effects of ABG-001 on the PKC, PLC, PKA, JNK, p38, Ras, Raf, and NGF Signaling Pathways and PC12 Cell Differentiation.

Because PKC is located downstream of PI3K, we investigated the function of PKC in ABG-001–induced neurite outgrowth using various PKC inhibitors (Gö6983, GF109203X, and Ro318220). Ro318220 and Gö6983 showed appreciable inhibitory effects (Fig. 6, A and B, Supplemental Fig. 1B). The percentage of neurite-bearing cells decreased from an initial value of 70.1 ± 2.8% to 36.0 ± 7.9% after treatment with Ro318220 (P < 0.01). Likewise, the percentage of neurite-bearing cells decreased from an initial value of 55.1 ± 3.0% to 23.0 ± 7.9% after treatment with Gö6983 (P < 0.05). Furthermore, a low concentration of NGF could augment the effects of ABG-001 on neurite outgrowth of PC12 cells (Fig. 6, C and D). These results show that PKC is involved in PC12 cell differentiation induced by ABG-001, but NGF signaling is not involved in the neurite outgrowth of PC12 cells induced by ABG-001.

In addition, inhibitors of PLC (U73122 and U73343), PKA (H-89), JNK (SP600125), p38 MAPK (SB203580), Raf (AZ628), and Ras (S3131 [sulindac sulfide], farnesylthiosalicylic acid) were used to investigate whether these signaling pathways are involved in neurite outgrowth mediated by ABG-001 in PC12 cells. The results showed that none of these inhibitors had a significant effect on the neurite outgrowth induced by ABG-001. These results verify that the signaling pathways associated with these proteins are not involved in the NGF-mimicking effects of ABG-001.

Discussion

ABG-001 (Fig. 1A) is a leading compound derived from the neuritogenic compound gentisides. It possesses a long alkyl chain of 14 carbons, two close hydroxy groups on the benzene ring, and an ester linkage between the ring and alkyl chain. Results from experiments aimed at investigating its structure-activity relationships showed that the length of the alkyl chain, number and position of hydroxyl groups on the benzene ring, and the type of linking group between the benzene ring and the alkyl chain had distinct effects on its neuritogenic activities (Luo et al., 2011).

NGF is known to induce both neurite outgrowth and survival by binding and phosphorylating the TrkA receptor in cell membranes (Chao, 2003). Therefore, we used the TrkA receptor inhibitor K252a to examine whether the TrkA signaling pathway is involved in the neurogenic effects of ABG-001. The results indicated that K252a had no inhibitory effect on the neurogenic function of ABG-001 (Fig. 1D; Supplemental Fig. 1A), which suggests that the TrkA receptor is not involved.

MEK and ERK1/2 have important functions in controlling gene transcription events leading to proliferation or differentiation of PC12 cells in response to NGF (Traverse et al., 1992; Rubinfeld and Seger 2005). NGF is known to promote PC12 cell survival via ERK1/2 signaling (Vaudry et al., 2002). In the present study, ABG-001 was confirmed to take part in neurite outgrowth of PC12 cells (Fig. 2, A–C) and to induce ERK and CREB phosphorylation (Fig. 2, D–G). Similar results were obtained when PD98059 [2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one] was used (Supplementary Fig. 2A). These results suggest that neurite outgrowth of PC12 cells after treatment with ABG-001 involves at least in part the MEK/ERK pathway. In addition, we found the early phase of CREB phosphorylation (Supplemental Fig. 5), but phosphorylation of ERK at the same time was not observed (unpublished data). At this point, it is possible that other signaling pathways take part in the regulation of ABG-001 NGF-mimicking effects.

The Ras/MAPK (Ras/ERK1/2), PLC-γ, and PI3K/Akt signaling pathways (Kaplan and Miller 1997; Kusunoki et al., 2008) are important for the neurogenic effects of NGF. Specific inhibitors of Ras, PLC, and PI3K/Akt, including S3131, farnesylthiosalicylic acid, U73122, U73343, LY294002, and wortmannin, were used in our study. Interestingly, only PI3K inhibitors were found to inhibit the neurogenic effects of ABG-001 (Fig. 3, A–C) and the phosphorylation of Akt by ABG-001 (Fig. 3, C and D). These results indicate that PI3K/Akt-mediated signaling plays an important role in the neurogenic effects of ABG-001.

Insulin/IGF-1 exerts important growth-promoting effects by activating the PI3K/Akt signaling pathway (Cui and Almazan 2007). The biologic actions of IGF-1 are mediated by the IGF-1 receptor, a member of the receptor tyrosine kinase family that induces dimerization and activates several downstream pathways to transmit proliferative signals after extracellular stimulations (Chitnis et al., 2008; Annenkov 2009; Bibollet-Bahena and Almazan 2009). Our results show that ABG-001 induces IGF-1 receptor phosphorylation and that AG1024, T9576, and IGF-1 receptor siRNA inhibit PC12 cell differentiation induced by ABG-001 (Figs. 4–5). These results suggest that the NGF-mimicking effects of IGF-1 on PC12 cells may be mediated via ABG-001 binding to IGF-1 receptor and thereby activating PI3K and MAPK signaling cascades to induce neuritogenic activities. However, PC12 cell differentiation induced by ABG-001 was not completely inhibited by AG1024, T9576, or siRNA against the IGF-1 receptor. This suggests that other receptors may also contribute to the NGF-mimicking effects of ABG-001.

Some studies have suggested that aside from the three signaling cascades described herein, the stress-activated protein kinase (SAPK)/JNK pathway also affects differentiation (Gelderblom et al., 2004). Therefore, we investigated the possible involvement of the SAPK/JNK pathway in regulating the NGF-mimicking effects of ABG-001 using a specific inhibitor SP600125 and the P38 inhibitor SB203580. Differentiation induced by ABG-001 was only weakly inhibited when PC12 cells were treated with SP600125 and SB203580 (unpublished data). These data suggest that the SAPK/JNK pathway does not participate in the neurogenic effects of ABG-001.

Previous reports also indicated that cAMP and PKC are involved in neuronal differentiation (Hansen et al., 2000; Kolkova et al., 2000). Therefore, the effects of cAMP-dependent protein kinase A (PKA) and PKC were investigated to explore other possible signaling pathways regulating the ABG-001–induced neurite outgrowth of PC12 cells. The inhibitory effects induced by PKC inhibitors were statistically significant (Fig. 6, A and B), but those induced by H-89, a PKA inhibitor, were not (unpublished data).

Taking these findings together, we propose that the neuritogenic effect of ABG-001 is mediated by a signaling cascade that follows this order: IGF-1 receptor, PI3K, Akt-PKC, ERK, CREB (Fig. 6, E and F).

Notably, the effective concentrations of some protein inhibitors (K252a, AG1024, T9576, and Ro318220) used in our study were higher than those described in other reports (Tapley et al., 1992; Alessi, 1997; Wen et al., 2001; Linder et al., 2007). In test experiments, we used the reported concentrations to investigate whether they have inhibitory effects on homologous proteins. These inhibitors did not produce inhibition of neurite outgrowth in PC12 cells (unpublished data). We note that a few other studies also used higher concentrations of these inhibitors (Yang and Wang, 2009; Pandya and Pillai, 2014). It is possible that differences in the purity or sources of inhibitors account for the apparent discrepancy in the concentrations at which these inhibitors are effective.

In conclusion, we demonstrated that ABG-001–induced time- and dose-dependent phosphorylation of IGF-1 receptor, Akt, ERK, and CREB in PC12 cells. Moreover, inhibition of IGF-1 receptor, PI3K, PKC, and ERK activation by AG1024, LY294002, Ro318220, and U0126 inhibitors, respectively, blocked the observed phosphorylation effects. In addition, siRNA against IGF-1 reduced the neurite outgrowth of PC12 cells induced by ABG-001 and IGF-1, like ABG-001, had NGF-mimicking effects. These results indicate that IGF-1 receptor-mediated MAPK activation is essential for ABG-001–induced neurite outgrowth in PC12 cells.

Authorship Contributions

Participated in research design: Qi, Osada.

Conducted experiments: Tang, Gao, Cao.

Contributed new reagents or analytic tools: Chen.

Wrote or contributed to the writing of the manuscript: Xiang, Kawatani.

Footnotes

    • Received January 7, 2015.
    • Accepted May 26, 2015.

  • This work was supported by the Natural Science Foundation of Zhejiang Province, People’s Republic of China [Grant Y2110105], the International Science and Technology Cooperation Program of China [No. 2014DFG32690], the National Natural Science Foundation of China [Grants 30873152 and 81072536], the Project for Science and Technology of Yunnan Province, China [Grant 2012AE002]. This work was inspired by the international and interdisciplinary environments of the JSPS Asian CORE Program, “Asian Chemical Biology Initiative.”

  • dx.doi.org/10.1124/mol.115.097758.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

ABG-001
tetradecyl 2,3-dihydroxybenzoate
AG1024
2-(3-bromo-5-(tert-butyl)-4-hydroxybenylidene)malononitrile
Akt
protein kinase B
AZ628
3-(2-cyanopropan-2-yl)-N-[4-methyl-3-[(3-methyl-4-oxoquinazolin-6-yl)amino]phenyl]benzamide
DMEM
Dulbecco’s modified Eagle’s medium
DMSO
dimethylsulfoxide
ERK
extracellular signal-regulated kinase
FAM
5-carboxy-fluorescein
GF109203X
bisindolylmaleimide I
Gö6983
3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione
H-89
N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide
HNMPA(AM)3
hydroxy-2-naphthalenylmethylphosphonic acid tris acetoxymethyl ester
JNK
c-Jun N-terminal kinase
K252a
(9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3ʹ,2ʹ,1ʹ-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one
LY294002
2-morpholin-4-yl-8-phenylchromen-4-one
MAPK
mitogen-activated protein kinase
MEK
mitogen-activated protein-extracellular signal-regulated kinase
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NGF
nerve growth factor
PD98059
2-(2-amino-3-methoxyphenyl)chromen-4-one
PI3K
phosphatidylinositol 3-kinase
PKA
protein kinase A
PKC
protein kinase C
PLC
phospholipase C
Ro318220
3-(1-(3-(amidinothio) propyl-1H-indol-3-yl))-3-(1-methyl-1H-indol-3-yl)maleimide
S3131
sulindac sulfide
SAPK
stress-activated protein kinase
SB203580
4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine
siRNA
small-interfering RNA
SP600125
1,9-pyrazoloanthrone
T9576
picropodophyllotoxin
TrkA
tyrosine kinase A
U0126
(2Z,3Z)-2,3-bis[amino-(2-aminophenyl)sulfanylmethylidene]butanedinitrile
U73122
1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione
U73343
1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione

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The Action Mechanism of ABG-001

Ruiqi Tang, Lijuan Gao, Makoto Kawatani, Jianzhong Chen, Xueli Cao, Hiroyuki Osada, Lan Xiang and Jianhua Qi
Molecular Pharmacology August 1, 2015, 88 (2) 326-334; DOI: https://doi.org/10.1124/mol.115.097758
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