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Effect of scoparone on neurite outgrowth in PC12 cells
Neuroscience Letters 440 (2008) 14–18
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of scoparone on neurite outgrowth in PC12 cells Yoo Jung Yang a , Hak Ju Lee b , Don Ha Choi b , Hai Shan Huang a , Sung Cil Lim a , Myung Koo Lee a,∗ a College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, Republic of Korea b Division of Wood Chemistry and Microbiology, Korea Forest Research Institute, Seoul 130-712, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 April 2008 Received in revised form 8 May 2008 Accepted 8 May 2008 Keywords: Scoparone Neurite outgrowth Cyclic AMP PC12 cells
a b s t r a c t The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 M, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 M also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 M scoparone. In addition, scoparone at 200 M increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+ /calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparoneinduced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells. © 2008 Published by Elsevier Ireland Ltd.
Neurotrophic factors, such as nerve growth factor (NGF), brainderived neurotrophic factor (BDNF) and fibroblast growth factor (FGF), play an important role in differentiation, survival and maintenance by stimulating neurite outgrowth in neuronal and PC12 cells [5,18], and they have therefore been considered for treating the neurodegenerative diseases. NGF induces microtubule biosynthesis to promote stable and long-term neurite outgrowth through its specific receptor tyrosine kinase (TrkA) [19]. TrkA receptor activation leads to neurite outgrowth via phosphorylation of extracellular signal-regulated protein kinase (ERK) by mitogen-activated protein kinase (MEK) in PC12 cells [19]. Similar to the effects of NGF, cyclic AMP can induce neuronal differentiation, such as neurite outgrowth, either on its own or via the activation of ERK and cyclic AMP-dependent protein kinase (PKA) in neuronal and PC12 cells [4,23]. Forskolin, an inducer of cyclic AMP formation, also induces neurite outgrowth in PC12 cells [8]. PKA activation leads to ERK activation [6]. In addition, the influx of calcium causes extensive neurite outgrowth through the initial activities of the Trk-Ras-ERK pathway, which is involved in cell differentiation [17]. Protein kinase C (PKC) and Ca2+ /calmodulin
∗ Corresponding author at: College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, 12 Gaeshin-dong, Heungduk-gu, Cheongju 361-763, Republic of Korea. Tel.: +82 43 261 2822; fax: +82 43 276 2754. E-mail address: [email protected] (M.K. Lee). 0304-3940/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2008.05.051
kinase II (CaMK II) also lead to cellular differentiation via ERK activation and thereby induce neurite outgrowth [1,13]. As a part of our ongoing search for neuronally active substances from natural resources, it has been found that the ethanol (EtOH) extract of the stem bark of Liriodendron tulipifera (Magnoliaceae) showed neurite outgrowth-promoting activity in PC12 cells. Therefore, the neurite outgrowth-induced bioactive substance was isolated as 6,7-dimethoxycoumarin (scoparone). Scoparone is a phytoalexin frequently produced in infected plant tissues [2]. Scoparone has also been proven to have potent antiinflammatory [11], free radical scavenging, immunosuppressive and vasodilating [9,10] activities. However, the effects of scoparone on neurite outgrowth have not been elucidated. Therefore, in the present study, we investigated the effects of scoparone on neurite outgrowth and its mechanism of intracellular signaling using rat adrenal pheochromocytoma (PC12) cells as a model for neuronal functions [5]. NGF (2.5S), H89, GF109203X, KN62 and 3-(4,5-dimethyl2-thiazolyl)-2,5-di-phenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). U0126 was purchased from Calbiochem (San Diego, CA, USA). RPMI 1640, fetal bovine serum (FBS), horse serum (HS) and antibiotics were obtained from Gibco (Carlsbad, CA, USA). All other chemicals were of reagent grade. PC12 cells were grown in RPMI medium 1640 supplemented with 10% HS, 5% FBS, 100 U/ml penicillin and 100 g/ml
Y.J. Yang et al. / Neuroscience Letters 440 (2008) 14–18
streptomycin and placed in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦ C [5]. The morphology of PC12 cells was observed as described previously [12]. PC12 cells were plated at low density (ca. 1 × 104 cells/cm2 ) onto poly-l-lysine-coated culture dishes. Neurite outgrowth was determined by manually tracing the length of the longest neurite per cell for a given 100 cells in a field using a phase-contrast microscope with 200× magnification. The numbers of differentiated cells were also determined by counting cells that had at least one neurite with a length of more than 1.5-fold of the diameter of the cell body, and they were expressed as a percentage relative to the positive control (30 ng/ml NGF, 100%) from six random-field observations of at least 100 cells. ERK activity was assayed as described previously [23]. Lysate aliquots (30 g) were fractionated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were probed with phosphor-ERK antibody (1:1000 dilution) and horseradish peroxidase-linked anti-rabbit IgG (Cell Signaling Technology, Beverly, MA, USA). The blots were then incubated for 1 min with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) and exposed to X-ray film. The levels of intracellular cyclic AMP were measured using an enzyme immunoassay system kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Intracellular Ca2+ concentrations were measured using a FLUO4/AM (4 M) (Molecular Probes, Eugene, OR, USA) and a confocal system (Ex. 488 nm; Em. 522 nm; LEICA Microsystems GmbH, Heidelberg, Germany) [7]. Traces deriving from total cell area were calculated as f/f0 , where f is the fluorescence emission of a single FLUO4/AM loaded cell at times ranging from 2 to x s, and f0 is the mean value of fluorescence intensities of the same cells calculated among all the images before the addition of the first stimulus. PKA, PKC and CaMK II activities were determined by enzymelinked immunosorbent assays, such as the non-radioactive PKA and PKC (Stressgen, Victoria, Canada) and CaMK II (CycLex Co., Nagano, Japan) activity assay kits. Cell viability was determined by conventional MTT assay using a Bauty Diagnostic Microplate Reader (570 nm, Molecular Devices, Sunnyvale, CA, USA) [16]. Protein content was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin. All data were presented as means ± S.E.M. of at least four experiments. Statistical analysis was performed using ANOVA followed by Tukey’s test. A voucher specimen of the stem bark of L. tulipifera has been deposited in the herbarium of the Division of Wood Chemistry and Microbiology, Korea Forest Research Institute (Seoul, Korea). Airdried stem bark of L. tulipifera (5 kg) were extracted with EtOH, and
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Fig. 2. Phase-contrast photomicrographs of PC12 cells after various treatments for 6 days. (A) Untreated control culture. (B) Culture treated with 200 M scoparone. (C) Culture treated with 30 ng/ml NGF. (D) Culture treated with 200 M scoparone in the presence of 2 ng/ml NGF. Scale bar = 50 m.
the EtOH extract was partitioned and chromatographed to obtain the bioactive substance scoparone (30 mg) according to the method described previously [14]. Scoparone was identified as a white powder: EI-MS m/z: 206 (M+ ); 1 H NMR (500 MHz, chloroform-d): ı 3.93 (3H, s, OMe-6), 3.96 (3H, s, OMe-7), 6.29 (1H, d, J = 9.5 Hz, H-3), 6.84 (1H, s, H-8), 6.87 (1H, s, H-5), 7.63 (1H, d, J = 9.5 Hz, H-4); 13 C NMR (125 MHz, chloroform-d): ı 56.60 (s, OMe-6), 56.62 (s, OMe-7), 100.28 (s, C-8), 108.34 (s, C-5), 111.69 (s, C-9), 113.77 (s, C-3), 143.50 (s, C-4), 146.63 (s, C-6), 150.29 (s, C-10), 153.14 (s, C-7), 161.60 (s, C-2); 1 H–1 H COSY correlations: H-4 ↔ H-3; HMBC correlations: H-3 → C-2/C-9, H-4 → C-2/C-5/C-10, H-5 → C-4/C-6/C-7/C-9/C-10, H-8 → C-6/C-7/C-9/C-10, OMe-6 → C-6, OMe-7 → C-7. The results of MS, 1 H NMR and 13 C NMR spectra, and the physical constants of scoparone were consistent with previous reports [14]. Scoparone at concentrations of 12.5–400 M induced neurite outgrowth from PC12 cells in a concentration-dependent manner in normal culture medium containing 10% HS and 5% FBS for a period of 6 days (Fig. 1A). Scoparone at 200 M resulted in neurite lengths of approximately 40 m per cell for a given 100 cells for 6 days (Fig. 1A). Under these culture conditions, 200 M scoparone also increased the steady outgrowth of neurites for 7 days (Fig. 1B). According to MTT assay, scoparone did not affect cell viability at concentrations up to 1 mM for 24 h in PC12 cells (data not shown).
Fig. 1. The concentration–response (A) and time course (B) curves for scoparone-induced neurite outgrowth in PC12 cells. Cells (ca. 1 × 104 cells/cm2 ) were treated with scoparone on 0, 2, 4 and 6 days. Neurite length for given 100 cells was quantified as the average radial distance from the neurite tips to the soma. Results represent means ± S.E.M. (n = 6) of four experiments. Significantly different from the control values: *P < 0.05; **P < 0.01 (ANOVA followed by Tukey’s test).
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Fig. 3. Effects of scoparone on phosphorylaton of ERK in PC12 cells. (A) PC12 cells were stimulated with scoparone (200 M) and harvested for Western blot analysis using the antibodies against phosphorylated ERK (top) and total ERK (bottom). (B) Relative density ratio in total ERK was expressed as 1 arbitrary unit. Results represent means ± S.E.M. of four experiments. Significantly different from control values: *P < 0.05; **P < 0.01 (ANOVA followed by Tukey’s test).
PC12 cells grown in normal culture medium were in round shape without neurite extension (Fig. 2A). At a concentration of 200 M alone, scoparone induced significant neurite elongation in PC12 cells (Fig. 2B). NGF at 2 ng/ml induced neurite outgrowth only a few cells, while 30 ng/ml NGF caused a robust outgrowth of neurites (Fig. 2C). The addition of NGF (30–50 ng/ml) resulted in neurite outgrowth in over 95% of PC12 cells for 6 days (data not shown). Scoparone at 200 M also enhanced NGF (2 ng/ml)-induced neurite outgrowth for 6 days (Fig. 2D). Microscopic observations indicated that scoparone and scoparone plus NGF resulted in the formation of long neurites, which extended to neighboring cells over distances of 50 m (Fig. 2B and D). These results indicated that scoparone could morphologically promote the differentiation of PC12 cells, resulting in neurite outgrowth. It is known that ERK activation induced by the stimulation of receptor TrkA [19] and the activation of PKA, PKC and CaMK II induced by cyclic AMP and an influx of Ca2+ [1,6,13] lead to neurite outgrowth in PC12 cells. Therefore, an analysis of the signaling pathways implicated in scoparone-induced differentiation was performed. As shown in Fig. 3A and B, scoparone at 200 M rapidly increased the phosphorylation of ERK by twofold in comparison to the control levels at 15 min. ERK phosphorylation was increased by 1.5–2-fold for 60 min. At concentrations of 50–300 M, scoparone increased the intracellular levels of cyclic AMP to 112–263% of the control levels at 15 min (Fig. 4A). The scoparone-induced increase in the levels of cyclic AMP was maintained for 30 min. Phosphorylated PKA was also increased to 2.8, 5.2 and 5.6 ng per mg protein by treat-
Fig. 4. Effects of scoparone on intracellular cyclic AMP levels (A) and PKA activity (B) in PC12 cells. (A) After treatments with scoparone (50–300 M) for 5 and 15 min at 37 ◦ C, the intracellular cyclic AMP levels were measured by using an enzyme immunoassay system kit. Cyclic AMP levels of control were 278.6 ± 15.2 pmol/mg protein. (B) After scoparone (200 M) were added to PC12 cells for 5–30 min, the cells were lysed and phosphorylated PKA from protein extracts (10 g) was analyzed. Further comments see Fig. 3.
Fig. 5. Effects of scoparone on intracellular Ca2+ concentration (A), and phosphorylation of PKC (B) and CaMK II (C) in PC12 cells. Scoparone (200 M) were added to PC12 cells. (A) The intracellular Ca2+ concentration of scoparone-responsive PC12 cells was performed using FLUO4/AM [8]. The data shown here are representative of a minimum of five independent experiments. Scoparone-treated cells were incubated for 5, 10 and 30 min (B) and for 30, 45 and 60 min (C). Phosphorylated PKC and CaMK II from protein extracts (10 or 3 g) were analyzed. Further comments see Fig. 3.
Y.J. Yang et al. / Neuroscience Letters 440 (2008) 14–18
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The occurrence of inflammatory events, such as pathogen invasion, trauma and stroke, can increase the production of reactive oxygen and nitrogen species within the central nervous system, which in turn contributes to the neurodegenerative processes. In the present study, scoparone not only exhibited neuritogenic activity but also potent anti-inflammatory and free radical scavenging activities [10,11]. Scoparone is also a phytoalexin [2] and may therefore play an important role in pathogen resistance. These data indicate that scoparone might have potential for application in the treatment of neurodegenerative diseases. In conclusion, the present study demonstrates that scoparone can markedly promote neurite outgrowth through amplification of the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells. The in vivo pharmacological effects of scoparone need further investigation. Fig. 6. Changes of PC12 cell differentiation by scoparone after exposure to various inhibitors. Cells were plated at ca. 1 × 104 cells/cm2 and were treated with U0126 (1 M), H89 (1 M), GF109203X (1 M), or KN62 (1 M) in the absence or presence of 2 ng/ml NGF. Cells with at least one neurite with a length longer than 1.5-fold of the diameter of the cell body were counted and expressed as a percentage of relative to the positive control (30 ng/ml NGF, 100%). Results represent means ± S.E.M. (n = 6) of four experiments. Significantly different from each control: a P < 0.01, compared to the untreated cells; b P < 0.01, compared to scoparone-treated cells; c P < 0.01, compared to NGF (2 ng/ml)-treated cells; d P < 0.01, compared to scoparone associated with NGF (2 ng/ml)-treated cells (ANOVA followed by Tukey’s test).
ment with 200 M scoparone at 5, 15 and 30 min, respectively (Fig. 4B). In addition, scoparone at 200 M significantly increased the intracellular concentrations of Ca2+ (Fig. 5A). Under the same conditions, phosphorylated PKC was increased to 6.0 g/mg protein at 5 min (Fig. 5B). Phosphorylated CaMK II was also increased to 27.5 mU at 60 min by treatment with 200 M scoparone (Fig. 5C). Next, the involvement of the ERK, cyclic AMP-PKA and Ca2+ releated secondary signaling pathways in the neuritogenic activity of scoparone were confirmed by using their inhibitors in PC12 cells. At a concentration of 200 M alone, scoparone significantly increased the neurite outgrowth-promoting activity by up to 77%. NGF at 2 ng/ml also induced neurite outgrowth to 44%, and the neurite outgrowth was enhanced to 87% by scoparone (200 M) associated with NGF (2 ng/ml) (Fig. 6). However, the increase in neurite outgrowth induced by 200 M scoparone was reduced by up to 47%, 40%, 21% and 32% by treatment with a specific MEK inhibitor (U0126, 1 M), a PKA inhibitor (H89, 1 M), a PKC inhibitor (GF109203X, 1 M) and a CaMK II inhibitor (KN62, 1 M), respectively (Fig. 6). These inhibitors also reduced the numbers of cells with neurite outgrowth induced by co-treatment with scoparone (200 M) and NGF (2 ng/ml). These results suggest that activation of the ERK, PKA, PKC and CaMK II signaling pathways is required for scoparone-induced neurite outgrowth in PC12 cells. It is difficult to understand the diverse signal transduction pathways of neurite outgrowth activated in response to NGF and their related cellular responses [3]. However, it has been suggested that the neuritogenic activity induced by scoparone was partially involved in the amplification of the upstream steps of ERK, PKA, PKC and CaMK II in the NGF receptor-mediated intracellular signaling pathway in PC12 cells. Several natural compounds, such as nardosinone (100 M) [15], sargaquinoic acid (1.5 g/ml) [20], a derivative of verbenachalcone (15 M) [24] and sargachromenol (30 M) [21], have been found to induce neurite outgrowth in PC12 cells. These compounds can also enhance neurite outgrowth in PC12 cells when co-treated with NGF. ␣-Phenyl-N-tert-butylnitron (10 mM) was recently found to induce neurite outgrowth from PC12 cells in the absence of NGF [22], which is effective at relatively higher concentrations than scoparone (200 M).
Acknowledgements The authors sincerely thank the Research Center for Bioresource and Health, ITEP and MKE for their financial support. References [1] S.T. Abraham, H.A. Benscoter, C.M. Schworer, H.A. Singer, A role for Ca2+ /calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells, Circ. Res. 81 (1997) 575–584. [2] G. Arras, G. D’Hallewin, M.G. Molinu, A. Dore, T. Venditti, M. Fois, G. Lima, M. Agabbio, Induction of phytoalexins biosynthesis in orange fruit by the biocontrol yeast Rhodotorula glutinis, Commun. Agric. Appl. Biol. Sci. 71 (2006) 915–921. [3] G. D’arcangelo, S. Halegoua, A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Raf-mediated gene inductions, Mol. Cell. Biol. 13 (1993) 3146–3155. [4] K. Freeland, Y.Z. Liu, D.S. Latchman, Distinct signaling pathways mediate the cAMP response element (CRE)-dependent activation of the calcitonin generelated peptide gene promoter by cAMP and nerve growth factor, Biochem. J. 345 (2000) 233–238. [5] L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A. 73 (1976) 2424–2428. [6] S.S. Grewal, A.M. Horgan, R.D. York, G.S. Withers, G.A. Banker, P.J. Stork, Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase, J. Biol. Chem. 275 (2000) 3722–3728. [7] S. Guarnieri, G. Fano, M.P. Rathbone, M.A. Mariggio, Cooperation in signal transduction of extracellular guanosine 5 -triphosphate and nerve growth factor in neuronal differentiation of PC12 cells, Neuroscience 128 (2004) 697–712. [8] T.O. Hansen, J.F. Rehfeld, F.C. Nielsen, Cyclic AMP-induced neuronal differentiation via activation of p38 mitogen-activated protein kinase, J. Neurochem. 75 (2000) 1870–1877. [9] H.C. Huang, Y.L. Huang, J.H. Chang, C.C. Chen, Y.T. Lee, Possible mechanism of immunosuppressive effect of scoparone (6,7-dimethoxycoumarin), Eur. J. Pharmacol. 217 (1992) 143–148. [10] H.C. Huang, C.R. Lee, Y.I. Weng, M.C. Lee, Y.T. Lee, Vasodilator effect of scoparone (6,7-dimethoxycoumarin) from a Chinese herb, Eur. J. Pharmacol. 218 (1992) 123–128. [11] S.I. Jang, Y.J. Kim, H.J. Kim, J.C. Lee, H.Y. Kim, Y.C. Kim, Y.G. Yun, H.H. Yu, Y.O. You, Scoparone inhibits PMA-induced IL-8 and MCP-1 production through suppression of NF-B activation in U937 cells, Life Sci. 78 (2006) 2937–2943. [12] Y. Kano, T. Nohno, R. Takahashi, T. Hasegawa, F. Hiragami, K. Kawamura, H. Motoda, T. Sugiyama, cAMP and calcium ionophore induce outgrowth of neuronal processes in PC12 mutant cells in which nerve growth factor-induced outgrowth of neuronal processes is impaired, Neurosci. Lett. 303 (2001) 21–24. [13] K. Kolkova, V. Novitskaya, N. Pedersen, V. Berezin, E. Bock, Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway, J. Neurosci. 20 (2000) 2238–2246. [14] S.H. Lee, B.K. Kim, S.H. Cho, K.H. Shin, Phytochemical constituents from the fruits of Acanthopanax sessiliflorus, Arch. Pharm. Res. 25 (2002) 280–284. [15] P. Li, K. Matsunaga, K. Yamamoto, R. Yoshikawa, K. Kawashima, Y. Ohizumi, Nardosinone, a novel enhancer of nerve growth factor in neurite outgrowth from PC12D cells, Neurosci. Lett. 273 (1999) 53–56. [16] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays, J. Immunol. Methods 65 (1983) 55–63. [17] T. Sakai, T. Furuyama, Y. Ohoka, N. Miyazaki, S. Fujioka, H. Sugimoto, M. Amasaki, S. Hattori, T. Matsuya, S. Inagaki, Mouse semaphorin H induces PC12 cell neurite
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of scoparone on neurite outgrowth in PC12 cells Yoo Jung Yang a , Hak Ju Lee b , Don Ha Choi b , Hai Shan Huang a , Sung Cil Lim a , Myung Koo Lee a,∗ a College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, Republic of Korea b Division of Wood Chemistry and Microbiology, Korea Forest Research Institute, Seoul 130-712, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 April 2008 Received in revised form 8 May 2008 Accepted 8 May 2008 Keywords: Scoparone Neurite outgrowth Cyclic AMP PC12 cells
a b s t r a c t The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 M, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 M also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 M scoparone. In addition, scoparone at 200 M increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+ /calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparoneinduced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells. © 2008 Published by Elsevier Ireland Ltd.
Neurotrophic factors, such as nerve growth factor (NGF), brainderived neurotrophic factor (BDNF) and fibroblast growth factor (FGF), play an important role in differentiation, survival and maintenance by stimulating neurite outgrowth in neuronal and PC12 cells [5,18], and they have therefore been considered for treating the neurodegenerative diseases. NGF induces microtubule biosynthesis to promote stable and long-term neurite outgrowth through its specific receptor tyrosine kinase (TrkA) [19]. TrkA receptor activation leads to neurite outgrowth via phosphorylation of extracellular signal-regulated protein kinase (ERK) by mitogen-activated protein kinase (MEK) in PC12 cells [19]. Similar to the effects of NGF, cyclic AMP can induce neuronal differentiation, such as neurite outgrowth, either on its own or via the activation of ERK and cyclic AMP-dependent protein kinase (PKA) in neuronal and PC12 cells [4,23]. Forskolin, an inducer of cyclic AMP formation, also induces neurite outgrowth in PC12 cells [8]. PKA activation leads to ERK activation [6]. In addition, the influx of calcium causes extensive neurite outgrowth through the initial activities of the Trk-Ras-ERK pathway, which is involved in cell differentiation [17]. Protein kinase C (PKC) and Ca2+ /calmodulin
∗ Corresponding author at: College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, 12 Gaeshin-dong, Heungduk-gu, Cheongju 361-763, Republic of Korea. Tel.: +82 43 261 2822; fax: +82 43 276 2754. E-mail address: [email protected] (M.K. Lee). 0304-3940/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2008.05.051
kinase II (CaMK II) also lead to cellular differentiation via ERK activation and thereby induce neurite outgrowth [1,13]. As a part of our ongoing search for neuronally active substances from natural resources, it has been found that the ethanol (EtOH) extract of the stem bark of Liriodendron tulipifera (Magnoliaceae) showed neurite outgrowth-promoting activity in PC12 cells. Therefore, the neurite outgrowth-induced bioactive substance was isolated as 6,7-dimethoxycoumarin (scoparone). Scoparone is a phytoalexin frequently produced in infected plant tissues [2]. Scoparone has also been proven to have potent antiinflammatory [11], free radical scavenging, immunosuppressive and vasodilating [9,10] activities. However, the effects of scoparone on neurite outgrowth have not been elucidated. Therefore, in the present study, we investigated the effects of scoparone on neurite outgrowth and its mechanism of intracellular signaling using rat adrenal pheochromocytoma (PC12) cells as a model for neuronal functions [5]. NGF (2.5S), H89, GF109203X, KN62 and 3-(4,5-dimethyl2-thiazolyl)-2,5-di-phenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). U0126 was purchased from Calbiochem (San Diego, CA, USA). RPMI 1640, fetal bovine serum (FBS), horse serum (HS) and antibiotics were obtained from Gibco (Carlsbad, CA, USA). All other chemicals were of reagent grade. PC12 cells were grown in RPMI medium 1640 supplemented with 10% HS, 5% FBS, 100 U/ml penicillin and 100 g/ml
Y.J. Yang et al. / Neuroscience Letters 440 (2008) 14–18
streptomycin and placed in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦ C [5]. The morphology of PC12 cells was observed as described previously [12]. PC12 cells were plated at low density (ca. 1 × 104 cells/cm2 ) onto poly-l-lysine-coated culture dishes. Neurite outgrowth was determined by manually tracing the length of the longest neurite per cell for a given 100 cells in a field using a phase-contrast microscope with 200× magnification. The numbers of differentiated cells were also determined by counting cells that had at least one neurite with a length of more than 1.5-fold of the diameter of the cell body, and they were expressed as a percentage relative to the positive control (30 ng/ml NGF, 100%) from six random-field observations of at least 100 cells. ERK activity was assayed as described previously [23]. Lysate aliquots (30 g) were fractionated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were probed with phosphor-ERK antibody (1:1000 dilution) and horseradish peroxidase-linked anti-rabbit IgG (Cell Signaling Technology, Beverly, MA, USA). The blots were then incubated for 1 min with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) and exposed to X-ray film. The levels of intracellular cyclic AMP were measured using an enzyme immunoassay system kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Intracellular Ca2+ concentrations were measured using a FLUO4/AM (4 M) (Molecular Probes, Eugene, OR, USA) and a confocal system (Ex. 488 nm; Em. 522 nm; LEICA Microsystems GmbH, Heidelberg, Germany) [7]. Traces deriving from total cell area were calculated as f/f0 , where f is the fluorescence emission of a single FLUO4/AM loaded cell at times ranging from 2 to x s, and f0 is the mean value of fluorescence intensities of the same cells calculated among all the images before the addition of the first stimulus. PKA, PKC and CaMK II activities were determined by enzymelinked immunosorbent assays, such as the non-radioactive PKA and PKC (Stressgen, Victoria, Canada) and CaMK II (CycLex Co., Nagano, Japan) activity assay kits. Cell viability was determined by conventional MTT assay using a Bauty Diagnostic Microplate Reader (570 nm, Molecular Devices, Sunnyvale, CA, USA) [16]. Protein content was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin. All data were presented as means ± S.E.M. of at least four experiments. Statistical analysis was performed using ANOVA followed by Tukey’s test. A voucher specimen of the stem bark of L. tulipifera has been deposited in the herbarium of the Division of Wood Chemistry and Microbiology, Korea Forest Research Institute (Seoul, Korea). Airdried stem bark of L. tulipifera (5 kg) were extracted with EtOH, and
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Fig. 2. Phase-contrast photomicrographs of PC12 cells after various treatments for 6 days. (A) Untreated control culture. (B) Culture treated with 200 M scoparone. (C) Culture treated with 30 ng/ml NGF. (D) Culture treated with 200 M scoparone in the presence of 2 ng/ml NGF. Scale bar = 50 m.
the EtOH extract was partitioned and chromatographed to obtain the bioactive substance scoparone (30 mg) according to the method described previously [14]. Scoparone was identified as a white powder: EI-MS m/z: 206 (M+ ); 1 H NMR (500 MHz, chloroform-d): ı 3.93 (3H, s, OMe-6), 3.96 (3H, s, OMe-7), 6.29 (1H, d, J = 9.5 Hz, H-3), 6.84 (1H, s, H-8), 6.87 (1H, s, H-5), 7.63 (1H, d, J = 9.5 Hz, H-4); 13 C NMR (125 MHz, chloroform-d): ı 56.60 (s, OMe-6), 56.62 (s, OMe-7), 100.28 (s, C-8), 108.34 (s, C-5), 111.69 (s, C-9), 113.77 (s, C-3), 143.50 (s, C-4), 146.63 (s, C-6), 150.29 (s, C-10), 153.14 (s, C-7), 161.60 (s, C-2); 1 H–1 H COSY correlations: H-4 ↔ H-3; HMBC correlations: H-3 → C-2/C-9, H-4 → C-2/C-5/C-10, H-5 → C-4/C-6/C-7/C-9/C-10, H-8 → C-6/C-7/C-9/C-10, OMe-6 → C-6, OMe-7 → C-7. The results of MS, 1 H NMR and 13 C NMR spectra, and the physical constants of scoparone were consistent with previous reports [14]. Scoparone at concentrations of 12.5–400 M induced neurite outgrowth from PC12 cells in a concentration-dependent manner in normal culture medium containing 10% HS and 5% FBS for a period of 6 days (Fig. 1A). Scoparone at 200 M resulted in neurite lengths of approximately 40 m per cell for a given 100 cells for 6 days (Fig. 1A). Under these culture conditions, 200 M scoparone also increased the steady outgrowth of neurites for 7 days (Fig. 1B). According to MTT assay, scoparone did not affect cell viability at concentrations up to 1 mM for 24 h in PC12 cells (data not shown).
Fig. 1. The concentration–response (A) and time course (B) curves for scoparone-induced neurite outgrowth in PC12 cells. Cells (ca. 1 × 104 cells/cm2 ) were treated with scoparone on 0, 2, 4 and 6 days. Neurite length for given 100 cells was quantified as the average radial distance from the neurite tips to the soma. Results represent means ± S.E.M. (n = 6) of four experiments. Significantly different from the control values: *P < 0.05; **P < 0.01 (ANOVA followed by Tukey’s test).
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Fig. 3. Effects of scoparone on phosphorylaton of ERK in PC12 cells. (A) PC12 cells were stimulated with scoparone (200 M) and harvested for Western blot analysis using the antibodies against phosphorylated ERK (top) and total ERK (bottom). (B) Relative density ratio in total ERK was expressed as 1 arbitrary unit. Results represent means ± S.E.M. of four experiments. Significantly different from control values: *P < 0.05; **P < 0.01 (ANOVA followed by Tukey’s test).
PC12 cells grown in normal culture medium were in round shape without neurite extension (Fig. 2A). At a concentration of 200 M alone, scoparone induced significant neurite elongation in PC12 cells (Fig. 2B). NGF at 2 ng/ml induced neurite outgrowth only a few cells, while 30 ng/ml NGF caused a robust outgrowth of neurites (Fig. 2C). The addition of NGF (30–50 ng/ml) resulted in neurite outgrowth in over 95% of PC12 cells for 6 days (data not shown). Scoparone at 200 M also enhanced NGF (2 ng/ml)-induced neurite outgrowth for 6 days (Fig. 2D). Microscopic observations indicated that scoparone and scoparone plus NGF resulted in the formation of long neurites, which extended to neighboring cells over distances of 50 m (Fig. 2B and D). These results indicated that scoparone could morphologically promote the differentiation of PC12 cells, resulting in neurite outgrowth. It is known that ERK activation induced by the stimulation of receptor TrkA [19] and the activation of PKA, PKC and CaMK II induced by cyclic AMP and an influx of Ca2+ [1,6,13] lead to neurite outgrowth in PC12 cells. Therefore, an analysis of the signaling pathways implicated in scoparone-induced differentiation was performed. As shown in Fig. 3A and B, scoparone at 200 M rapidly increased the phosphorylation of ERK by twofold in comparison to the control levels at 15 min. ERK phosphorylation was increased by 1.5–2-fold for 60 min. At concentrations of 50–300 M, scoparone increased the intracellular levels of cyclic AMP to 112–263% of the control levels at 15 min (Fig. 4A). The scoparone-induced increase in the levels of cyclic AMP was maintained for 30 min. Phosphorylated PKA was also increased to 2.8, 5.2 and 5.6 ng per mg protein by treat-
Fig. 4. Effects of scoparone on intracellular cyclic AMP levels (A) and PKA activity (B) in PC12 cells. (A) After treatments with scoparone (50–300 M) for 5 and 15 min at 37 ◦ C, the intracellular cyclic AMP levels were measured by using an enzyme immunoassay system kit. Cyclic AMP levels of control were 278.6 ± 15.2 pmol/mg protein. (B) After scoparone (200 M) were added to PC12 cells for 5–30 min, the cells were lysed and phosphorylated PKA from protein extracts (10 g) was analyzed. Further comments see Fig. 3.
Fig. 5. Effects of scoparone on intracellular Ca2+ concentration (A), and phosphorylation of PKC (B) and CaMK II (C) in PC12 cells. Scoparone (200 M) were added to PC12 cells. (A) The intracellular Ca2+ concentration of scoparone-responsive PC12 cells was performed using FLUO4/AM [8]. The data shown here are representative of a minimum of five independent experiments. Scoparone-treated cells were incubated for 5, 10 and 30 min (B) and for 30, 45 and 60 min (C). Phosphorylated PKC and CaMK II from protein extracts (10 or 3 g) were analyzed. Further comments see Fig. 3.
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The occurrence of inflammatory events, such as pathogen invasion, trauma and stroke, can increase the production of reactive oxygen and nitrogen species within the central nervous system, which in turn contributes to the neurodegenerative processes. In the present study, scoparone not only exhibited neuritogenic activity but also potent anti-inflammatory and free radical scavenging activities [10,11]. Scoparone is also a phytoalexin [2] and may therefore play an important role in pathogen resistance. These data indicate that scoparone might have potential for application in the treatment of neurodegenerative diseases. In conclusion, the present study demonstrates that scoparone can markedly promote neurite outgrowth through amplification of the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells. The in vivo pharmacological effects of scoparone need further investigation. Fig. 6. Changes of PC12 cell differentiation by scoparone after exposure to various inhibitors. Cells were plated at ca. 1 × 104 cells/cm2 and were treated with U0126 (1 M), H89 (1 M), GF109203X (1 M), or KN62 (1 M) in the absence or presence of 2 ng/ml NGF. Cells with at least one neurite with a length longer than 1.5-fold of the diameter of the cell body were counted and expressed as a percentage of relative to the positive control (30 ng/ml NGF, 100%). Results represent means ± S.E.M. (n = 6) of four experiments. Significantly different from each control: a P < 0.01, compared to the untreated cells; b P < 0.01, compared to scoparone-treated cells; c P < 0.01, compared to NGF (2 ng/ml)-treated cells; d P < 0.01, compared to scoparone associated with NGF (2 ng/ml)-treated cells (ANOVA followed by Tukey’s test).
ment with 200 M scoparone at 5, 15 and 30 min, respectively (Fig. 4B). In addition, scoparone at 200 M significantly increased the intracellular concentrations of Ca2+ (Fig. 5A). Under the same conditions, phosphorylated PKC was increased to 6.0 g/mg protein at 5 min (Fig. 5B). Phosphorylated CaMK II was also increased to 27.5 mU at 60 min by treatment with 200 M scoparone (Fig. 5C). Next, the involvement of the ERK, cyclic AMP-PKA and Ca2+ releated secondary signaling pathways in the neuritogenic activity of scoparone were confirmed by using their inhibitors in PC12 cells. At a concentration of 200 M alone, scoparone significantly increased the neurite outgrowth-promoting activity by up to 77%. NGF at 2 ng/ml also induced neurite outgrowth to 44%, and the neurite outgrowth was enhanced to 87% by scoparone (200 M) associated with NGF (2 ng/ml) (Fig. 6). However, the increase in neurite outgrowth induced by 200 M scoparone was reduced by up to 47%, 40%, 21% and 32% by treatment with a specific MEK inhibitor (U0126, 1 M), a PKA inhibitor (H89, 1 M), a PKC inhibitor (GF109203X, 1 M) and a CaMK II inhibitor (KN62, 1 M), respectively (Fig. 6). These inhibitors also reduced the numbers of cells with neurite outgrowth induced by co-treatment with scoparone (200 M) and NGF (2 ng/ml). These results suggest that activation of the ERK, PKA, PKC and CaMK II signaling pathways is required for scoparone-induced neurite outgrowth in PC12 cells. It is difficult to understand the diverse signal transduction pathways of neurite outgrowth activated in response to NGF and their related cellular responses [3]. However, it has been suggested that the neuritogenic activity induced by scoparone was partially involved in the amplification of the upstream steps of ERK, PKA, PKC and CaMK II in the NGF receptor-mediated intracellular signaling pathway in PC12 cells. Several natural compounds, such as nardosinone (100 M) [15], sargaquinoic acid (1.5 g/ml) [20], a derivative of verbenachalcone (15 M) [24] and sargachromenol (30 M) [21], have been found to induce neurite outgrowth in PC12 cells. These compounds can also enhance neurite outgrowth in PC12 cells when co-treated with NGF. ␣-Phenyl-N-tert-butylnitron (10 mM) was recently found to induce neurite outgrowth from PC12 cells in the absence of NGF [22], which is effective at relatively higher concentrations than scoparone (200 M).
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