Vol. 38, No. 2169 Biol. Pharm. Bull. 38, 169–178 (2015)

Regular Article

DA-9801 Promotes Neurite Outgrowth via ERK1/2-CREB Pathway in PC12 Cells Jong Hoon Won,a Kyong Hoon Ahn,a Moon Jung Back,a Hae Chan Ha,a Ji Min Jang,a Ha Hyung Kim,b Sang-Zin Choi,c Miwon Son,c and Dae Kyong Kim*,a a

 Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University; 221 Huksuk-dong, Dongjak-gu, Seoul 156–756, South Korea: b Biotherapeutics and Glycomics Laboratory, College of Pharmacy, ChungAng University; 221 Huksuk-dong, Dongjak-ku, Seoul 156–756, South Korea: and c Phytomedicine & Functional Food Research, Pharmaceutical Product Research Laboratories, Research Center of Dong-A ST Co., Ltd.; 21 Geumhwa-ro, 105 Beon-gil, Giheung-gu, Yongin-si, Gyeonggi-do 446–905, South Korea. Received March 17, 2014; accepted November 19, 2014; advance publication released online December 5, 2014 In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy. Key words DA-9801; diabetic peripheral neuropathy; neurite outgrowth; extracellular signal-regulated kinase 1/2; cAMP response element-binding protein (CREB)

Peripheral neuropathy is a common disease in diabetes patients. According to recent estimates, more than half of all diabetes patients develop diverse neuropathic symptoms.1,2) Diabetic peripheral neuropathy (DPN) is one of the most debilitating complications of Type 1 and Type 2 diabetes, and its histopathology is characterized by neuronal small fiber degeneration, demyelination, and atrophy.3–5) Hyperglycemia, a metabolic disruption known to increase oxidative stress in the peripheral nerves, is an important factor in the pathogenesis of DPN.6) In patients with diabetes, as well as those with local nerve compression that needs to be treated to prevent or repair permanent nerve damage, treatment of the underlying cause might result in partial or full pain relief.7) Nerve growth factor (NGF) itself has been considered as an option for the treatment of DPN. NGF restores the nociceptive threshold for thermal noxious stimuli,8) normalizes myelinated nerve fiber morphology,9) and reduces neurogenic vasodilatation10) in streptozotocin (STZ)-induced diabetic rats. Unfortunately, a recently concluded phase III trial failed to demonstrate the efficacy of NGF administration.11) The reasons for this failure include its limited delivery to the nervous system and an unwanted apoptotic effect elicited through the interaction with the p75NTR receptor.11–13) Additionally, the effectiveness of NGF application as a therapeutic intervention to treat brain injury is limited by the numerous unwanted

side effects observed in both animals and patients, reflecting the diversity of cell types that react to NGF, both centrally and peripherally.14–16) To avoid the side effects, development of orally active small molecules that potentiate the expression and activity of NGF is warranted.17,18) The extracts of plants from the Dioscorea genus were reported to exhibit hypoglycemic,19) immunostimulatory,20) and anti-inflammatory effects,21) as well as anti-tumor22) and anti-osteoporotic activity.23) Dioscorea japonica THUNBERG. (DJ) has been utilized in the treatment of hyperglycemia in Korea.24,25) Treatment with the extract of Dioscorea rhizome was shown to increase endogenous NGF levels in the salivary gland and sciatic nerve in mice.18) DA-9801 is an extract obtained from a mixture of DJ and Dioscorea nipponica MAKINO (DN). While the rhizome extracts showed a significant effect on neurite outgrowth and Trk-A phosphorylation in neurons, it remains to be determined whether DA-9801 possesses these neurotrophic properties.18,25) NGF is known to interact with two receptor proteins, TrkA and p75NTR.26,27) NGF signaling through TrkA elicits many of the classical neurotrophic actions ascribed to NGF.28) Binding of NGF stimulates the autophosphorylation of TrkA and induces a signal transduction cascade involving phosphoinositide 3-kinase (PI3K), extracellular-signal-regulated kinase (ERK), and Akt, leading to survival, regeneration, and

 To whom correspondence should be addressed.  e-mail: [email protected] *  © 2015 The Pharmaceutical Society of Japan

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differentiation of neurons.29–31) In PC12 cells, proteins of the Raf family32–34) mediate NGF signaling through phosphorylation (and thereby activation) of the dual-specificity mitogenactivated protein (MAP) kinase kinase (MEK1).32,35,36) MEK1 activation leads to the phosphorylation of two members of the MAP kinase family, ERK1/2.37,38) ERK1/2 are phosphorylated on threonine 202 and tyrosine 204 residues by MEK1,39) leading to activation and translocation of ERK1/2 into the nucleus.40) Additional transcription factors contribute to the regulation of c-fos transcription in response to NGF signaling. The cAMP regulatory element binding protein (CREB) is a transcription factor that binds to the cAMP response element (CRE) site within the c-fos promoter (Berkowitz et al. 1989). NGF signaling leads to the phosphorylation of CREB at serine 133 via a Ras-dependent mechanism.41) In the present study, our aim was to examine the effect of DA-9801 on the promotion of neurite outgrowth in PC12 cells and elucidate the underlying mechanisms. Since several signal transduction molecules have been implicated in NGFmediated induction of neurite outgrowth,42) we evaluated the effects of specific inhibitors of the ERK1/2 cellular signaling pathway on the enhancement of this particular effect of NGF by DA-9801. Our results indicate that DA-9801 potentiates the neuritogenesis-stimulating action of NGF through the ERK1/2-CREB signaling pathway.

MATERIALS AND METHODS Materials RPMI-1640, fetal bovine serum (FBS), horse serum (HS), and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from GIBCO (Grand Island, NY, U.S.A.). Biocoat poly-D -lysine-coated 6-, 24-, and 96-well microplates and 100-mm dishes were purchased from Becton Dickinson (Bedford, MA, U.S.A.). NGF was purchased from Enzo Life Sciences (Farmingdale, NY, U.S.A.). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). GW5074 and PD98059 were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). DA-9801 was graciously provided by Dong-A Pharmaceutical Company (Yong-in, Korea). DA-9801 was dissolved in sterile water to obtain a 50 mg/mL stock solution. Anti-glyceraldehyde3-phosphate dehydrogenase (anti-GAPDH) antibody was purchased from Bethyl Laboratories (Montgomery, TX, U.S.A.). Anti-p42/44, anti-phospho-p42/44, anti-CREB, and anti-phospho-CREB antibodies were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Extra pure grade methanol and ethanol was purchased from Duksan (Ansansi, Kyounggido, Korea). Ultrapure Tris and EDTA disodium dihydrate was purchased from Duchefa (Haarlem, the Netherlands). GW5074, PD98059, SL327 and SB203580 were from cayman chemical (Ann Arbor, MI, U.S.A.). Unless otherwise stated, all reagents used in this study were purchased from Sigma. Preparation of Rhizome Extract of DJ and DN The detailed description of methods of extraction was previously published.18) Dried DJ and DN were purchased at a specialized market for traditional herbal medicine (Kyungdong herb market, Seoul, Korea) and their identity was confirmed by Dr. Changsoo Yuk (a specialist in plant classification, Kyung Hee University). The voucher specimen was deposited at Dong-A Pharmaceutical (Youngin-shi, Korea). The rhizome mixture of DJ and DN (1 : 3, 30 kg) was extracted with 50% ethanol at

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room temperature for 48 h, and concentrated under vacuum. The total yield of the extract (DA-9801) was 10.0%. HPLC analysis was performed for the quantitative determination of dioscin and allantoin, the marker components of DJ and DN, in DA-9801. DA-9801 was found to contain both dioscin (0.414%) and allantoin (1.218%).18) The chromatographic analysis was carried out on a Luna C18 column (Phenomenes, Torrace, CA, U.S.A.) at room temperature with a linear gradient elution of a methanol–water mobile phase (3 : 7 to 9 : 1, v/v) at a flow rate of 1.0 mL/min. The analysis time was less than 40 min. Cell Culture The rat pheochromocytoma cell line, PC12, was obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). PC12 cells were maintained on poly-D -lysine-coated plates in RPMI-1640 supplemented with 10% horse serum and 5% heat-inactivated FBS. PC12 cells between passages 5 and 20 were cultured and cells were grown at 37°C in a humidified 5% CO2 air atmosphere. DA-9801’s Effect on PC12 Cell Viability PC12 cell viability was determined using CCK-8. PC12 cells were plated in poly-D -lysine-coated 96-well plates at a density of 3×104 cells per well in RPMI-1640 containing 10% HS and 5% FBS, and incubated for 1 d. The cells were incubated with DA-9801 in the presence or absence of NGF for 72 h. NGF was dissolved in sterile water at a stock concentration of 10 µg/mL. RPMI-1640 medium containing reduced serum levels (2% HS and 1% FBS) was used for drug treatment. CCK solution was added to each well and incubated for 4 h. Absorbance was measured using a microplate reader (BioTeK Synergy H1 Hybrid Reader) at a wavelength of 450 nm. Evaluation of PC12 Cell Neurite Outgrowth PC12 cells were plated in poly-D -lysine-coated 24-well plates in RPMI-1640 containing 10% HS and 5% FBS at a density of 1.5×104 cells per well. After overnight incubation, the medium was changed to RPMI-1640 with 2% HS and 1% FBS. The cells were treated with DA-9801 either alone or in combination with other agents, in the presence or absence of NGF, for 72 h. GW5074, PD98059, SL327 and SB203580 also added. All inhibitors were dissolved in dimethyl sulfoxide (DMSO) with the final concentration of DMSO not exceeding 0.1%. The cell images were captured using Motic Images Plus 2.0 software (Motic Instruments Inc., Richmond, Canada). Cells with outgrowths longer than the cell body diameter were scored positive for neurites and their number was expressed as a percentage of the total cell number. RNA Isolation and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, U.S.A.). For real-time qPCR, 0.1 µg of total RNA was reverse transcribed using a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). PCR was performed in triplicate using the MyiQ Single-Color RealTime PCR Detection System and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, U.S.A.). PCR amplification was performed using the Bio-Rad CFX Connect Real-Time System (Bio-Rad, Hercules, CA, U.S.A.) for 40 cycles. Thermal cycling conditions included pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 57°C for 15 s, and extension at 72°C for 20 s. The primer sequences used were as follows: GAPDH, forward (5′-TGC ACC ACC AAC TGC TTA G-3′) and reverse (5′-GGA TGC AGG GAT GAT GTT C-3′);

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neurofilament-L (NF-L), forward (5′-AGA CAT CAG CGC CAT GCA-3′) and reverse (5′-TTC GTG CTT CGC AGC TCA T-3′). Relative expression was normalized to GAPDH levels. Western Blot Analysis Cells were homogenized by sonication at 0°C in homogenization buffer (25 m M Tris, 1.25 m M EDTA, and 0.1% Triton X-100, pH 7.5 (Amresco, Solon, OH, U.S.A.)) containing Protease Inhibitor Cocktail and PhosStop (Roche Diagnostics, Mannheim, Germany). Lysates were centrifuged at 2000×g at 4°C for 10 min, and the supernatant was collected. Proteins in the lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. (GE Healthcare, Bio-Science, NJ, U.S.A.). The membrane was blocked for 1 h by using Tris-buffered saline containing 5% bovine serum albumin (BSA) (Bioworld, Dublin, OH, U.S.A.) and 0.1% Tween 20 (Amresco). Immunoblots were performed with anti-phospho-ERK1/2 (Thr202/Tyr204; 1 : 1000 dilution), anti-ERK1/2 (1 : 1000), GAPDH (1 : 1000), anti-phospho-CREB (Ser133), and anti-CREB antibodies (Cell Signaling Technology, Beverly, MA, U.S.A.) overnight at 4°C in the same buffer. Membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit or antimouse immunoglobulin G (IgG) (Cell Signaling Technology) for 2 h, and proteins were detected using the Enhanced Luminol Chemiluminescence system (GE Healthcare, Bio-Science,

Fig. 1.

NJ, U.S.A.). Statistical Analysis All data are expressed as means±S.E.M. Statistical differences between groups were analyzed by oneway ANOVA with subsequent Tukey’s tests. In all cases, p