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P2X1 receptor-mediated Ca2+ influx triggered by DA-9801 potentiates nerve growth factor-induced neurite outgrowth Moon Jung Back, Hae Kyung Lee, Joo Hyun Lee, Zhicheng Fu, Mi Won Son, SangZin Choi, Hyo Sang Go, Sungjae Yoo, Sun Wook Hwang, and Dae Kyong Kim ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00082 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 23, 2016

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ACS Chemical Neuroscience

P2X1 receptor-mediated Ca2+ influx triggered by DA-9801 potentiates nerve growth factor-induced neurite outgrowth

Moon Jung Back†, Hae Kyung Lee†, Joo Hyun Lee†, Zhicheng Fu†, Mi Won Son‡, Sang Zin Choi‡, Hyo Sang Go‡, Sungjae Yoo§, Sun Wook Hwang§ and Dae Kyong Kim*,†

†

Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang

University; 84, Heukseok-ro, Dongjak-Ku, Seoul 06974, Republic of Korea ‡

Department of Research Planning & Management, Research Center of Dong-A ST Co., Ltd.;

21 Geumhwa-ro, 105 beon-gil, Giheung-gu, Yongin-si, Gyeonggi-do, 446-905, Republic of Korea §

Department of Biomedical Sciences, Korea University College of Medicine, Seoul 136-705,

Republic of Korea *

Corresponding author

Tel: 82-2-820-5610; Fax: 82-2-825-7920; E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. The increase in intracellular calcium ion level was induced by DA9801,

and

chelation

of

extracellular

calcium

ions

with

ethylene-

bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Keywords: P2X1, Calcium ion influx, Neurite outgrowth, DA-9801


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Introduction Nerve growth factor (NGF) is a neurotrophic factor, which facilitates neuronal differentiation and maintains neuronal survival.1,

2

In various neuronal disorders such as

Parkinson's disease (PD)3, diabetic neuropathy4-6, and chemo-induced peripheral neuropathy (CIPN)7, NGF levels markedly decrease. Therefore, NGF administration was suggested to treat neurodegenerative diseases. The protective effects of NGF on Alzheimer's disease (AD)8-10, PD11, diabetic neuropathy12, 13, and CIPN14-17 were established in experimental models. However, NGF failed to demonstrate the efficacy in phase III clinical trials for diabetic neuropathy.18 The reason for the failure was the occurrence of undesirable side effects at the applied dose of NGF. Thus, a therapeutic strategy to enhance trophic effects of NGF is needed. A recent phase II clinical study of DA-9801, a well-standardized Dioscorea japonica Thunb. and Dioscorea nipponica Makino extract, was completed in the USA, showing that DA-9801 effectively alleviated pain in the patients. Owing to the known potentiating effects of DA-9801 on NGF19, a study of the mechanism underlying the effect of DA-9801 is required for developing a strategy to enhance the NGF trophic effect. Our previous study showed that the potentiation effects of DA-9801 on neurite outgrowth were mediated by the extracellular-signal-regulated kinase1/2 (ERK1/2)/cyclic AMP response element-binding protein (CREB) pathway.20 However, the mechanism by which DA-9801 enhances ERK1/2 signaling requires further investigation, and the molecular targets of DA-9801 should be identified. P2 receptors have emerged as regulators of neuronal development, from neuritogenesis to functional maturation.21 Both subfamilies of P2 receptors, P2X receptors (P2X1–P2X7) and P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–P2Y14), are



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commonly expressed in neurons.21 The roles of P2X and P2Y receptors in neuronal functions have been widely investigated.22-24 However, the roles of the individual P2 receptor subtypes in neuronal differentiation remain largely unknown. In the present study, we revealed, for the first time, the role of the P2X1 receptor in the potentiation of NGF-induced neurite outgrowth through a mechanism study of DA-9801. We showed that calcium ion influx through the P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via phosphorylation of ERK1/2. These findings may provide a novel therapeutic target for neurodegeneration-associated diseases.

Results 1. Calcium ion influx induced by DA-9801 potentiates NGF-induced neuritogenesis Before investigating DA-9801 molecular targets, we confirmed that DA-9801 enhanced neurite outgrowth, a hallmark of neuronal differentiation25-27, in the presence of a low concentration of NGF (2 ng/mL) in PC12 cells (Figure 1A). There is much evidence showing that neuronal axon growth is modulated by changes in the intracellular calcium ion level.28 To investigate whether the calcium ion level is involved in the potentiation of neuritogenesis by DA-9801, we first analyzed the change in intracellular calcium ion level by DA-9801 treatment using Fluo-4 NW dye. The increase in intracellular calcium ion level by 30 µg/mL DA-9801 treatment for 15 min was observed by fluorescence microscopy (Figure 1B, left). Treatment with a higher level of DA-9801 (100 µg/mL) induced a large increase in intracellular calcium level in the presence or absence of NGF within 200 sec (Figure 1B, right). To explore whether the calcium ion influx induced by DA-9801 affects neurite outgrowth, PC12 cells were pretreated with the extracellular calcium chelator ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) prior to DA-9801 exposure. As shown in


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Figure 1C, the increased percentage of neurite-bearing cells elicited by DA-9801 was significantly reduced by the EGTA treatment. The data indicated that calcium ion influx was required for the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. 2. Calcium ion influx participates in DA-9801-induced ERK1/2 phosphorylation ERK1/2 play a major role in neurite outgrowth by mediating the downstream signaling of the NGF-activated tropomyosin receptor kinase A (TrkA) receptor.29 We confirmed that DA-9801 enhanced the phosphorylation of ERK1/2 with or without NGF in a dose-dependent manner (Figure 2A). Moreover, the neurite outgrowth-promoting effect of DA-9801 was inhibited by the ERK kinase inhibitor PD98059 (Figure 2B), suggesting that ERK mediates the effect of DA-9801. To evaluate whether calcium ion influx affects ERK phosphorylation, the effect of calcium chelation with EGTA on the DA-9801-induced ERK1/2 phosphorylation was assessed by an immunoblotting assay. As shown in Figure 2C, calcium chelating with EGTA attenuated the DA-9801-induced ERK1/2 phosphorylation. Interestingly, treatment with a TrkA receptor inhibitor, GW441756, had little effect on the ERK1/2 phosphorylation evoked by DA-9801 (Figure 2D). The data suggest that the calcium ion influx is an upstream event of DA-9801-induced ERK1/2 phosphorylation. 3. P2X1 mediates DA-9801-induced ERK1/2 phosphorylation To determine which calcium ion-permeable channel mediates the calcium ion influx triggered by DA-9801, the levels of the DA-9801-induced ERK1/2 phosphorylation were examined in the presence of various calcium-channel inhibitors (Table 1)30-35,57-58. Among these inhibitors, the P2 receptor inhibitor pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), at 10 µM, significantly reduced the ERK1/2 phosphorylation induced by DA9801 (Figure 3A, left). Interestingly, the reducing effect of PPADS on DA-9801-induced ERK1/2 phosphorylation was not shown at a concentration of 100 µM (Figure 3A, right).



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Treatment with the other inhibitors had little effect on DA-9801-induced ERK1/2 phosphorylation (Figure 3B). The different effects of different concentrations of PPADS may be attributable to the diverse inhibitory potency of PPADS on P2 receptor subunits. The IC50 values of PPADS at P2X1, P2X3, P2Y1, and P2Y13 are lower than the concentration (10 µM) reducing the ERK1/2 phosphorylation elicited by DA-9801.30, 31, 36 The IC50 of PPADS at the other P2 receptor subunits is much higher than 10 µM, or PPADS does not act on these subunits. 30, 31, 36

Therefore, effects of subtype-specific antagonists for P2X1, P2X3, P2Y1, and P2Y13 on

the DA-9801-induced ERK1/2 phosphorylation were investigated. The IC50 values of the P2 subtype-specific antagonists are indicated in Table 2.37-40 As shown in Figures 4A and 4B, both NF449 (P2X1 antagonist) and NF110 (P2X1 and P2X3 antagonist) significantly attenuated the DA-9801-induced ERK1/2 phosphorylation. MRS2179, a P2Y1 antagonist, had little effect on the levels of ERK1/2 phosphorylation (Figure 4C). MRS2211 (P2Y13 antagonist) treatment seemed to decrease the DA-9801-induced phosphorylation of ERK1/2, but the decrease was not statistically significant (Figure 4D). Taken together, the results suggest that P2X1, the common target of NF449 and NF110, contributes to the DA-9801induced ERK1/2 phosphorylation. 4. P2X1 receptor mediates the potentiation effect of DA-9801 on NGF-induced neurite outgrowth In addition to the effect of P2X1 on ERK1/2 phosphorylation, we investigated whether P2X1 actually contributes to the neurite outgrowth-promoting effect of DA-9801. To measure the neurite outgrowth more precisely, neurite lengths were determined using the Neurite Tracer plugin for ImageJ, which traces β-tubulin III-positive neurites. We found that increased neurite lengths evoked by DA-9801 was significantly reduced by NF449 treatment


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(Figures 5A and 5B). However, treatment with another P2 subtype antagonist MRS2211, which seemed to attenuate DA-9801-induced ERK1/2 phosphorylation (p = 0.137), did not suppress the DA-9801-enhanced neurite outgrowth (Figure 5C). Furthermore, the DA-9801enhanced neurite outgrowth was suppressed by knockdown of P2X1 using siRNA (Figure 6), suggesting a role for P2X1 in promoting neuritogenesis. To appraise whether DA-9801 directly activates P2X1, the patch-clamp analysis in heterologous expression systems expressing P2X1 was performed. Using whole-cell voltage clamp experiments with mouse P2X1-expressing HEK293 cells, no significant increase in the electrical current upon exposure to DA-9801 was recorded in the cells that showed P2X1mediated currents in response to α,β-methylene ATP, a P2X-selective agonist (Supplementary Figure S1). Therefore, P2X1 may be a final effector of DA-9801-induced responses in PC12 cells, but it seems to involve an intracellular signal transduction rather than the direct binding of the components of DA-9801 to the P2X1 receptor.

Discussion Neuroregeneration has been considered a strategy for overcoming degenerative neuronal disorders, including neuropathy associated with diabetes or chemotherapy.2 Neuroregeneration depends on neurite formation to restore injured neuronal synaptic connections.41 The outgrowth of neurites is induced and promoted by NGF.42 Thus, NGF plays an essential role in neuroregeneration. Indeed, a loss of NGF has been observed in patients with diabetic neuropathy4 or CIPN7, and reduced NGF immunoreactivity was observed in the basal forebrain complex of AD patients.43 A decreased serum level of NGF was reported in early-stage PD patients.3 Moreover, the NGF supply has therapeutic or preventive effects on experimental models of this neuronal disease.10, 11, 13, 14, 16, 17 However, a



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phase III clinical trial with subcutaneous administration of recombinant human (rh) NGF to 1,019 patients failed to demonstrate benefits because the dose of rhNGF was limited by the occurrence of adverse effects such as hyperalgesia, myalgias, and arthralgias.18 Therefore, potentiation of the trophic activity of NGF is required, rather than direct injection of the trophic factor. In this respect, understanding the molecular-based mechanism of action of DA-9801, which has a synergistic effect with NGF, is needed. DA-9801 is a well-standardized botanical drug for diabetic neuropathy. Enhanced NGF-induced neurite outgrowth caused by DA-9801 was observed in dorsal root ganglion neurons and neuroblastoma PC12 cells.19 Administration of DA-9801 improved the neuropathy in murine type 1 and type 2 diabetic experimental models.44-46 Moreover, the therapeutic effect of DA-9801 on diabetic pain was proven in recently completed phase II clinical trials in the USA. In the present study, we explored a molecular mechanism of the potentiation effect of DA-9801 on NGF-induced neurite outgrowth. Interestingly, DA-9801 induced an increase in the intracellular calcium concentration (Figure 1B). The role of the intracellular calcium signal as a modulator of neuronal axon growth has been widely studied.28 Our results showing that the increased neurite outgrowth elicited by DA-9801 was significantly reduced by the calcium chelator EGTA (Figure 1C) suggest that the calcium ion influx caused by DA9801 mediates its NGF-promoting effect. Since different calcium sources have diverse effects on neurite outgrowth,47, 48 the route of the calcium ion influx induced by DA-9801 must be identified. Among the various calcium-channel inhibitors tested, only PPADS, a P2 receptor inhibitor, significantly reduced the DA-9801-induced ERK1/2 phosphorylation (Figure 3A), which suggested that a P2 receptor may mediate the effect of DA-9801. The treatment with MK-801, an N-methyl-D-aspartate receptor (NMDAR) inhibitor, seemed to attenuate DA-


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9801-induced ERK1/2 phosphorylation, although the effect was not statistically significant (Figure 3B). The NMDAR may possibly be involved in the effect of DA-9801 and this possibility would be investigated further. Since seven subunit clones of P2X receptors and eight subunit clones of P2Y receptors have been identified in the P2 receptor family, we investigated which P2 receptor subtype mediates the neurotrophic effect of DA-9801. Among the subtype-specific antagonists, NF449 (a P2X1 antagonist) and NF110 (P2X1 and P2X3 antagonist) significantly reduced the ERK1/2 phosphorylation induced by DA-9801 (Figures 4A and 4B). Although the result from the NF110-treated experiment implies the possibility of the contribution of P2X3 to the DA-9801 effect, P2X1 was investigated because P2X1 is the common target of the two antagonists (NF449 and NF110), and the reduction levels of DA9801-induced ERK1/2 phosphorylation by these antagonists were similar. NF449 actually suppressed the neurite outgrowth-promoting effect of DA-9801 (Figures 5A and 5B). Furthermore, the DA-9801-enhanced neurite outgrowth was attenuated by transfection with siRNA of P2X1 (Figure 6). The results suggest that the P2X1 receptor mediates the NGFpromoting effect of DA-9801. P2X1 expression was detected in neonatal and adult rat brains by in situ hybridization,49 and increased P2X1 mRNA levels were observed during maturation of cerebellar granule neurons.50 In addition, increased expression of P2X1 was observed in survived neurons after hemicerebellectomy.51 Nevertheless, the role of P2X1 in neuronal regeneration has not been studied yet. In the present study, we revealed the contribution of P2X1 to neurite outgrowth for the first time. The P2X1 receptor recognizes extracellular ATP molecules with a high sensitivity.30 ATP levels increase in extracellular fluid after brain injury, such as inflammation, ischemia, and trauma.52 Similar to DA-9801, ATP has potentiation



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effects on NGF-induced neurite outgrowth.53, 54 Furthermore, a previous study showed that ATP activated ERK1/2, a key mediator of neurotrophic effects, and the pattern was parallel with that of DA-9801.54 In conclusion, the present study demonstrated that P2X1 receptor-mediated calcium ion influx triggered by DA-9801 potentiated the NGF-induced neurite outgrowth. The results obtained in this study imply a possible relationship between P2X1 and neuroregeneration. Although further studies are required to elucidate which component of DA-9801 affects P2X1 and whether additional targets of DA-9801 exist, the results suggest that P2X1 can be a useful candidate for a therapeutic target in neurodegeneration. Thus, the study may pave the way for the development of P2X1-targeting drugs for the treatment of neurodegenerative diseases.

Materials and Methods 1. Reagents DA-9801 is a Dioscorea rhizome extract produced by Dong-A ST Co. (Yong-in, Korea) as previously described. D. japonica and D. nipponica (in a ratio of 1 to 3) were extracted with 50% ethanol at room temperature (RT) for 48 h, and the extract was concentrated under vacuum. A voucher specimen was deposited at the Dong-A ST research center. The marker components of DA-9801 are dioscin (0.414%) and allantoin (1.218%), and were previously analyzed by HPLC.19 DA-9801 was dissolved in sterile water to obtain a 50 mg/mL stock solution. RPMI-1640

medium,

horse

serum

(HS),

fetal

bovine

serum

(FBS),

penicillin/streptomycin, an Alexa-488-conjugated anti-mouse immunoglobulin G (IgG)


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antibody, 4',6-diamidino-2-phenylindole (DAPI), ProLong Gold, Lipofectamine RNAiMAX, and Fluo-4 NW calcium assay kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Poly-D-lysine-coated 6- and 24-well plates, black 96-well plates, and 100-mm dishes were supplied by Corning (Corning, NY, USA). Poly-D-lysine-coated coverslips were obtained from Neuvitro (Vancouver, WA, USA). NGF was provided by Enzo Life Sciences (Farmingdale, NY, USA). All inhibitors (Tables 1 and 2) were obtained from Tocris Bioscience (Bristol, UK). Protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail were purchased from Roche (Basel, Switzerland). Bovine serum albumin (BSA) was obtained from Bioworld (Dublin, OH, USA). An anti-β-tubulin III antibody was supplied by EMD Millipore (Billerica, MA, USA). Anti-phospho-ERK1/2 (Thr202/Tyr204) and antiERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Alkaline phosphatase (AP)-conjugated anti-rabbit IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Tween-20 was provided by Amresco (Solon, OH, USA). Dimethyl sulfoxide (DMSO), nitro blue tetrazolium chloride (NBT), and 5-bromo-4chloro-3'-indolyl phosphate p-toluidine salt (BCIP) were supplied by Duchefa (Haarlem, Netherlands). Unless otherwise stated, all other reagents used in this study were purchased from Sigma–Aldrich (St. Louis, MO, USA). PD98059, GW441756, nifedipine, and flunarizine were dissolved in DMSO, with the final concentration of DMSO not exceeding 0.1%. The other agents were dissolved in sterile water or phosphate-buffered saline (PBS).

2. Cell culture The rat neuroblastoma PC12 cell line, which is widely used as a cellular model for studies of neurotrophic action25-27, was purchased from American Type Culture Collection



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(Manassas, VA, USA). PC12 cells were cultured in RPMI-1640 medium containing 10% HS, 5% FBS, and 100 U/mL of penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 in air.

3. Analysis of neurite outgrowth For counting neurite-bearing cells, PC12 cells were seeded in poly-D-lysine-coated 24-well plates at a density of 1.5 × 104 cells per well in the growth medium for one day. The medium was changed to a low-serum medium (2% HS and 1% FBS), and cells were treated with the indicated concentrations of DA-9801 for 72 h in the presence of 2 ng/mL of NGF. Cells were co-treated or pretreated with inhibitors or EGTA for the indicated periods. The groups that were not treated with inhibitors received the same volume of each vehicle. At the end of the treatment, phase-contrast images of cells were captured using the Motic Images Plus 2.0 software (Motic Instruments, Inc., Richmond, Canada). Only cells that had at least one neurite with a length equal to the cell body diameter were counted as neurite-bearing cells, and their number was expressed as a percentage of the total cell number. For more deliberate measuring of neurite outgrowth, neurite lengths were detected using automatic quantitative analysis. Cells were seeded in 4-well plates, which contained poly-D-lysine-coated coverslips, at a density of 1.5 × 104 cells per well in the growth medium for one day. The treatment was the same as above. At the end of the treatment, the cells were fixed with 4% formaldehyde for 20 min. The fixed cells were washed three times with 0.3% Triton X-100 (TX-100) in PBS and incubated with 5% BSA and 0.3% TX-100 in PBS for 1 h at RT. The cells were then incubated with the mouse monoclonal anti-β-tubulin III antibody (1:350) in 1% BSA, 0.3% TX-100 in PBS for 24 h at 4 °C. After three washes with 0.3% TX100 in PBS, the cells were incubated with the Alexa-488-conjugated anti-mouse antibody


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(1:350) for 1 h at RT. After three washes with 0.3% TX-100 in PBS, the cells were stained with DAPI and mounted with ProLong Gold. Images of cells were acquired with a Ni-U fluorescence microscope (Nikon, Tokyo, Japan) equipped with a digital camera (DS-Ri1, Nikon). The lengths of β-tubulin III-positive neurites were measured by Neurite Tracer for more than 100 cells per group of experiments.

4. Analysis of intracellular calcium ion levels For cytoplasmic calcium ion level measurements, the Fluo-4 NW calcium assay kit was used according to the manufacturer’s instructions. PC12 cells were seeded in poly-Dlysine-coated black 96-well plates (2.5 × 104 cells/well) and incubated for 24 h at 37 °C in an atmosphere of 5% (v/v) CO2. Then, the cells were incubated with a Fluo-4 NW dye solution at 37 °C for 30 min, followed by incubation at RT for an additional 30 min. Compounds were added to the assay plate, and the fluorescence was monitored with a FlexStation 3 microplate reader (494 nm excitation; 516 nm emission) (Molecular Devices, Sunnyvale, CA, USA). To image intracellular calcium ion level, PC12 cells were seeded in poly-D-lysinecoated coverslips (1.5 × 104 cells) in the growth medium for one day. The cells were incubated with a Fluo-4 NW dye solution under the same condition as above. The cells were then treated with or without 30 µg/mL DA-9801 for 15 min. Images of the green fluorescent dye and DIC (differential interference contrast) were obtained by a Ni-U fluorescence microscope.

5. Immunoblot analysis PC12 cells (1 × 106 cells/well) were seeded in a poly-D-lysine-coated 6-well plate in



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RPMI-1640 containing 10% HS and 5% FBS for 24 h, and then the medium was replaced with a low serum medium (1% HS and 0.5% FBS) for 24 h. The cells were then treated with the indicated concentrations of DA-9801 for 15 min in the presence or absence of 2 ng/mL of NGF. For inhibitor treatments, the cells were pretreated with the indicated concentrations of inhibitors (Tables 1 and 2) or EGTA for 3 h prior to the 30 µg/mL DA-9801 challenge for 15 min. The groups that were not treated with inhibitors received the same volume of each vehicle. At the end of the treatment, the cells were homogenized by sonication in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor and PhosSTOP phosphatase inhibitor cocktails. The lysates were centrifuged at 10,000 × g at 4 °C for 10 min, and the supernatants were collected. Proteins in the lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked for 1 h using Tris-buffered saline (TBS) containing 5% BSA and 0.1% Tween20 and then incubated with the anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 antibodies (1:1,000) in 5% BSA, 0.1% Tween-20 in TBS for 24 h at 4 °C. After three washes with 0.1% Tween-20 in TBS, the membranes were incubated with AP-conjugated anti-rabbit IgG (1:1,000) in 5% BSA, 0.1% Tween-20 in TBS for 1 h at RT. Proteins were detected using the chemical reaction of the NBT and BCIP substrates with AP. The ImageJ software was used to quantify the density of the bands. The levels of phospho-ERK1/2 were normalized to those of the total ERK1/2.

6. siRNA Transfection and RT-PCR PC12 cells were seeded in 4-well plates, which contained poly-D-lysine-coated coverslips, at a density of 1.5 × 104 cells per well in the growth medium for one day. The


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cells were then transfected with 50 nM ON-TARGETplus SMARTpool rat P2rx1 (the gene encoding P2X1 receptor) siRNA or 50 nM ON-TARGETplus non-targeting pool siRNA (Dharmacon, Lafayette, CO, USA) using Lipofectamine RNAiMAX according to the manufacturer’s instructions. After 30 h of transfection, the treatment was the same as neurite length measuring process. To confirm the expression and knockdown of the P2X1 receptor, the cells were seeded in poly-D-lysine-coated 6-well plates (5 × 105 cells/well) in the growth medium for 24 h, followed by transfection with P2rx1 siRNA or negative control siRNA as mentioned above. mRNA levels of P2rx1 and Hprt1 (the housekeeping gene) were analyzed by RT-PCR (reverse transcription polymerase chain reaction). Primer sequences used for the amplification of the P2rx155 and Hprt156 were as follows: Rat P2rx1, forward, 5′– ATG TTC TCC TGC AGG CCC AG-3′ and reverse, 5′– GTG CAG AAT GGG ACA AAC CG-3′; rat Hprt1, forward, 5′– TGT TTG TGT CAT CAG CGA AAG TG-3′ and reverse, 5′– ATT CAA CTT GCC GCT GTC TTT TA-3′. The MaximeTM i-Taq PCR PreMix (iNtRON Biotechnology, Seongnam, Republic of Korea) was used under the following PCR conditions: Five min of initial denaturation at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 60°C, and 1 min at 72°C. The PCR products were detected by ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA) after polyacrylamide gel electrophoresis.

7. Statistical analysis All data were expressed as the mean ± standard error of the mean (SEM). Differences between the groups were analyzed using a two-tailed Student’s t-test. In all cases, a p-value of < 0.05 was considered statistically signiﬁcant.



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Legends Figure 1. Calcium ion influx triggered by DA-9801 potentiated NGF-induced neurite outgrowth. (A) Concentration-dependent NGF-potentiating effect of DA-9801. PC12 cells were treated with various concentration of DA-9801 for 72 h in the presence or absence of a low concentration of NGF (2 ng/mL), and the percentage of neurite-bearing cells was assessed. The data represent mean ± SEM of three independent experiments. The statistical comparison of each DA-9801 untreated control vs. DA-9801 treated group (3, 10 or 30 µg/mL) was evaluated by unpaired Student’s t-test (significant differences, *p < 0.05). (B) The increased intracellular calcium ion level evoked by DA-9801. The green fluorescent Fluo-4 dye and DIC images were captured after treatment with or without 30 µg/mL DA-9801 for 15 min in PC12 cells (left). The red box in the 100× images was enlarged in 400× images (scale bar = 100 µm). DA-9801 alone (100 µg/mL) or in combination with NGF (2 ng/mL) was added to the PC12 cells at 20 s after (indicated by arrow) leading fluorescence (excitation 494 nm, emission 516 nm) using the FlexStation 3 system (right). (C) Suppression of the DA-9801 effect on neurite outgrowth by extracellular calcium chelating. PC12 cells were pretreated for 1 h with 500 µM EGTA prior to exposure to 30 µg/mL of DA-9801 for 72 h in the presence of NGF (2 ng/mL). The data represent the mean ± SEM of three independent experiments. Significant differences between the indicated groups, *p < 0.05 (unpaired Student’s t-test); N.S., not significant.

Figure 2. Calcium ion influx contributed to the DA-9801-induced ERK1/2


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phosphorylation. (A) Concentration-dependent ERK1/2 phosphorylation induced by DA-9801. PC12 cells were treated with 3 to 30 µg/mL of DA-9801 for 15 min in the presence or absence of 2 ng/mL of NGF. Statistical comparison of each DA-9801 untreated control vs. DA-9801 treated group (3, 10 or 30 µg/mL) was evaluated by paired Student’s t-test (significant differences, **p < 0.01 and ***p < 0.001). (B) Suppression of the DA-9801 effect on neurite outgrowth by the ERK kinase inhibitor PD98059. PC12 cells were challenged with 30 µg/mL of DA-9801 for 72 h in the presence of 2 ng/mL of NGF, with or without 10 µM PD98059, and neurite-bearing cells were counted. These data represent mean ± SEM of three independent experiments. Significant differences between the indicated groups, *p < 0.05 (unpaired Student’s t-test) (C) Reduction of DA-9801-induced ERK1/2 phosphorylation by calcium chelating. (D) A TrkA inhibitor showed little effect on DA-9801-induced ERK1/2 phosphorylation. PC12 cells were pretreated with or without each agent (2 mM EGTA, 1 µM GW441756, or the vehicle) for 3 h and then incubated with 30 µg/mL of DA-9801 for 15 min. Phospho-ERK1/2 and ERK1/2 were analyzed by immunoblotting assays. The data represent the mean ± SEM of three independent experiments. Significant differences between the indicated groups, *p < 0.05 and **p < 0.01 (paired Student’s t-test); N.S., not significant.

Figure 3. P2 receptor mediated the DA-9801-induced ERK1/2 phosphorylation. (A) Attenuation of the DA-9801-induced ERK1/2 phosphorylation by the P2 receptor inhibitor PPADS at a concentration of 10 µM. (B) Effects of other calcium-channel inhibitors on DA-9801-induced ERK1/2 phosphorylation. PC12 cells were pretreated with each agent (10 µM PPADS, 100 µM PPADS, 5 µM nifedipine, 20 µM ruthenium red, 10 µM flunarizine, 10 µM mecamylamine, 1 µM MK-801, or the vehicle) for 3 h prior to exposure to 30 µg/mL



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of DA-9801 for 15 min. Phospho-ERK1/2 and ERK1/2 were analyzed by immunoblotting assays. The data represent the mean ± SEM of three independent experiments. Significant differences between the indicated groups, *p < 0.05 and **p < 0.01 (paired Student’s t-test); N.S., not significant.

Figure 4. P2X1 facilitated the DA-9801-induced ERK1/2 phosphorylation. (A) Selective antagonists of P2 receptor subtypes. (B) Effect of the P2X1- and P2X3-subtype antagonist NF110 (100 nM) on DA-9801-induced ERK1/2 phosphorylation. (C) Effect of the selective P2X1 antagonist NF449 (1 nM) on DA-9801-induced ERK1/2 phosphorylation. (D) Effect of the selective P2Y1 antagonist MRS2179 (1 µM) on DA-9801-induced ERK1/2 phosphorylation. (E) Effect of the selective P2Y13 antagonist MRS2211 (1 µM) on DA9801-induced ERK1/2 phosphorylation. PC12 cells were pretreated with each antagonist for 3 h prior to exposure to 30 µg/mL of DA-9801 for 15 min. Phospho-ERK1/2 and ERK1/2 were analyzed by immunoblotting assays. The data represent the mean ± SEM of three independent experiments. Significant differences between the indicated groups, *p < 0.05, **p < 0.01, and ***p < 0.001 (paired Student’s t-test); N.S., not significant.

Figure 5. P2X1 receptor mediated the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. Suppression of the DA-9801 effect on neurite outgrowth by the P2X1 antagonist NF449. PC12 cells were pretreated with 1 nM NF449, 1 µM MRS2211, or each vehicle for 1 h and then incubated with 30 µg/mL of DA-9801 for 72 h in the presence of 2 ng/mL of NGF. The cells were stained with the anti-β-tubulin III antibody and DAPI post fixation with 4%


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formaldehyde. (A) Representative images of β-tubulin III-positive cells were acquired using a fluorescence microscope and skeletonized using the Neurite Tracer program. The arrows indicate neurites (scale bar = 100 µm). (B) Neurite lengths were digitally quantified using the Neurite Tracer program. The data represent the mean ± SEM of four independent experiments. Significant differences between the indicated groups, *p < 0.05 and **p < 0.01 (paired Student’s t-test); N.S., not significant.

Figure 6. Role of the P2X1 receptor in the promoting effect of DA-9801 on neuritogenesis confirmed by siRNA-mediated knockdown PC12 cells were transfected with 50 nM siRNA of rat P2X1 (siP2rx1) or 50 nM non-targeting negative control siRNA (siCON). After 30 h, cells were incubated with 30 µg/mL of DA9801 for 72 h in the presence of 2 ng/mL of NGF. Cells were stained with the anti-β-tubulin III antibody and DAPI post fixation with 4% formaldehyde. (A) Representative images of βtubulin III-positive cells were acquired using a fluorescence microscope and skeletonized using the Neurite Tracer program. Arrows indicate neurites (scale bar = 100 µm). (B) Neurite lengths were digitally quantified using the Neurite Tracer program. These data represent mean ± SEM of three independent experiments. Significant differences between the indicated groups, *p < 0.05 and **p < 0.01 (paired Student’s t-test); N.S., not significant. (C) RT-PCR expression analysis of siRNA-transfected PC12 cells with PCR primers specific for P2rx1 (predicted product size: 296 bp) or Hprt1 (predicted product size: 66 bp).

Table 1. Inhibitors of various calcium ion-permeable channels. Table 2. Selective antagonists of P2 receptor subtypes.



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Supporting Information Patch-clamp analysis in a heterologous expression system expressing the P2X1 receptor.

Acknowledgment This study was supported by the Chung-Ang University Excellent Student Scholarship and the Ministry of Trade, Industry and Energy R&D program (Grant no. 10039303).

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P2X1 receptor-mediated Ca2+ influx triggered by DA-9801 potentiates nerve growth factor-induced neurite outgrowth Moon Jung Back†, Hae Kyung Lee†, Joo Hyun Lee†, Zhicheng Fu†, Mi Won Son‡, Sang Zin Choi‡, Hyo Sang Go‡, Sungjae Yoo§, Sun Wook Hwang§ and Dae Kyong Kim*,† † Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang University; 84, Heukseok-ro, Dongjak-Ku, Seoul 06974, Republic of Korea ‡ Department of Research Planning & Management, Research Center of Dong-A ST Co., Ltd.; 21 Geumhwa-ro, 105 beon-gil, Giheung-gu, Yongin-si, Gyeonggi-do, 446-905, Republic of Korea § Department of Biomedical Sciences, Korea University College of Medicine, Seoul 136-705, Republic of Korea *Corresponding author Tel: 82-2-820-5610; Fax: 82-2-825-7920; E-mail: [email protected]

For Table of Contents Use Only


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A

C 18

18 w/o NGF NGF 2 ng/mL

16 14

*

Neurite bearing cells (%)


12 10 8 6 4

*

2 0

0

5

10 15 20 25 DA-9801 (μg/mL)

∗

∗

16 14 12

N.S.

10 8 6 4 2

0 EGTA DA-9801

30

− −

+ −

− +

+ +

B DA-9801

−

+

100X

DIC 100X

Fluo-4 400X

3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8

Intracellualar calcium (fold)

Fluo-4

Intracellualar calcium (fold)

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NT DA-9801

0

50

100

150

200

Time (secs)

Fig.1


3 2.8 2.6 2.4 2.2 2 1.8 1.6 1.4 1.2 1 0.8

NGF NGF + DA-9801

0

50

100

150

Time (secs)

200


A

B w/o NGF 0

3

10

30

P-ERK1/2

P-ERK1/2

ERK1/2

ERK1/2

5

3.0

P-ERK/ERK (Relative intensity)

∗∗

4

NGF (2 ng/mL)

DA-9801 (μg/mL)

3 2 1 0

0

3

10

30


DA-9801 (μg/mL)


∗∗∗

2.5 2.0 1.5 1.0 0.5 0.0

C

∗

∗

16 14 12 10 8 6 4 2 0

N.S.

PD98059

−

+

−

+

DA-9801

−

−

+

+

D EGTA

−

+

−

+

GW441756

−

+

−

+

DA-9801

−

−

+

+

DA-9801

−

−

+

+

P-ERK1/2

P-ERK1/2

ERK1/2

ERK1/2

8

∗∗

6 4

10

∗∗ P-ERK/ERK (Relative intensity)

10 P-ERK/ERK (Relative intensity)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

N.S.

2

6 4 2 0

0

Fig.2 ACS Paragon Plus Environment

∗

8

N.S.

N.S.

Page 31 of 36

A PPADS (10 μM) DA-9801

−

+

−

+

−

−

+

+

PPADS (100 μM) DA-9801

P-ERK1/2

P-ERK1/2

ERK1/2

ERK1/2

∗

∗∗

5 4 3

N.S.

2

−

+

−

+

−

−

+

+



6

1

N.S.

∗∗

4 3

∗

2 1

0

0

B Nifedipine DA-9801

− −

+ −

− +

+ +

Ruthenium red DA-9801

− −

+ −

− +

P-ERK1/2

P-ERK1/2

ERK1/2

ERK1/2

ERK1/2

3

10

N.S.

2 1

∗

8 6

N.S.

N.S.

4 2 0

0

Mecamylamine DA-9801

− −

+ −

− +

+ +

MK-801 DA-9801 P-ERK1/2

ERK1/2

ERK1/2

4 3 2 1

N.S.

N.S.

+ −



∗

− −

4 3 2 1 0

0

∗


N.S.

− +

+ +

N.S.

6

∗∗

5 4

N.S.

3 2 1 0

P-ERK1/2

5

+ −


4

N.S.

∗∗


5

− −

Flunarizine DA-9801

+ +

P-ERK1/2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


− +

+ +

p=0.07


A

B

NF449

−

+

−

+

NF110

−

+

−

+

DA-9801

−

−

+

+

DA-9801

−

−

+

+

P-ERK1/2

P-ERK1/2

ERK1/2

ERK1/2

6 4

N.S.

2

∗

∗∗



∗∗∗

∗∗

8

6 N.S.

4 2 0

0

D

C MRS2179

−

+

−

+

MRS2211

−

+

−

+

DA-9801

−

−

+

+

DA-9801

−

−

+

+

P-ERK1/2

P-ERK1/2

ERK1/2

ERK1/2

4 3 2

∗∗

N.S.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

N.S.

1 0

∗∗∗

3 2 1 0


N.S.

N.S.

Page 33 of 36


A NF449

−

+

−

+

DA-9801

−

−

+

+

β-tubulin III

skeletons of neurites

B 2.0

∗

1.5

C

∗

2.5 Neurite lengths (fold change)

Neurite lengths (fold change)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N.S.

1.0

0.5

N.S.

∗∗

2.0 N.S.

1.5 1.0 0.5 0.0

0.0 NF449

−

+

−

+

DA-9801

−

−

+

+

MRS2211

−

+

−

+

DA-9801

−

−

+

+



A siCON

+

−

+

−

siP2rx1

− −

+ −

− +

+ +

DA-9801 β-tubulin III

skeletons of neurites

B

C

∗

1.8 Neurite lengths (fold change)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

∗∗

1.6 1.4

N.S.

1.2 1.0

(bp)

0.8

P2rx1 300 200 Hprt1 75 50

0.6 0.4 0.2 0.0 siCON

+

−

+

−

siP2rx1

−

+

−

+

DA-9801

−

−

+

+


N rx1 2 siP

O

siC

Page 35 of 36

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ligand-gated calcium ion-permeable receptor Type

Inhibitor

P2 receptor AChR NMDAR TRPV

PPADS tetrasodium salt28, 29 Mecamylamine57 MK-80158 Ruthenium red30

Voltage-gated calcium ion channel Type

Inhibitor

L-type P-type N-type T-type

Nefedipine31, Ruthenium red32 Ruthenium red32 Ruthenium red32 Flunarizine33

Table 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Selective P2 receptor antagonists Type

Antagonist (IC50 in μM)

P2X1 P2X3 P2Y1 P2Y13

NF449 (0.0003)37, NF110 (0.08)38 NF110 (0.04)38 MRS2179 (0.33)39 MRS2211 (1.07)40

Table 2


Page 36 of 36

P2X1 Receptor-Mediated Ca2+ Influx Triggered by ... - ACS Publications

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