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2 signaling pathway
Neuroscience Letters 504 (2011) 121–126
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Visfatin induces neurite outgrowth in PC12 cells via ERK1/2 signaling pathway Young-Soon Kang a , Moon-Kyoung Bae b,e , Jee-Young Kim c , Joo-Won Jeong d , Il Yun a,e , Hye-Ock Jang a,e , Soo-Kyung Bae a,e,f,∗ a
Department of Dental Pharmacology, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea Department of Oral Physiology, School of Dentistry, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea Department of Physiology, School of Medicine, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea d Department of Anatomy and Neurobiology, School of Medicine, Kyunghee University, Seoul 130-701, South Korea e Research Institute for Oral Biotechnology, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea f Medical Research Institute, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea b c
a r t i c l e
i n f o
Article history: Received 21 July 2011 Received in revised form 25 August 2011 Accepted 11 September 2011 Keywords: CoCl2 ERK1/2 Neurite outgrowth PC12 cells Visfatin
a b s t r a c t The angiogenic and inflammatory functions of visfatin and its effect on vascular cells, are fairly well known. However, its role within the nervous system remains largely unclear. To gain insight into this area, we studied the neuritogenic effect of visfatin on PC12 rat pheochromocytoma cells. We investigated whether visfatin gene expression, which is upregulated by hypoxia in cancer cells, is associated with neuritogenesis in PC12 cells. Using RT-PCR, Western blot analysis, ELISA, morphological observations, and immunostaining, we initially showed that CoCl2 , a hypoxic mimetic agent, upregulated visfatin gene expression along with neurite outgrowth in PC12 cells. We also showed that visfatin stimulated neurite outgrowth in PC12 cells. Moreover, in PC12 cells, visfatin evoked the activation of the extracellular signalregulated kinase 1/2 (ERK1/2), which is closely linked to neuritogenesis. Visfatin-induced outgrowth of neurites was prevented by inhibition of the ERK1/2 pathway. Taken together, our results demonstrate for the first time that visfatin induces neurite outgrowth in PC12 cells via the activation of an ERK-dependent pathway, and suggest that visfatin may exert various biological, physiological, and pathological functions in not only the vascular system but also the nervous system. © 2011 Elsevier Ireland Ltd. All rights reserved.
Precise regulation of neurite outgrowth is critical for axonal growth in neurogenesis and provides the fundamental structure involved in neuronal transmission [8]. PC12 cells, derived from a rat pheochromocytoma, are a well-established model for studying neurite outgrowth; the first report on PC12 cells described neurite outgrowth by nerve growth factor (NGF) [11]. PC12 cells treated with neurotrophic factors, such as NGF, brain derived neurotrophic factor (BDNF), and fibroblast growth factor (FGF), cease division, extend neurites, and differentiate [8,13,32]. This process is largely mediated through TrkA, the high-affinity NGF receptor, leading to the activation of the extracellular signal-related kinase 1/2 (ERK1/2) pathway [14]. Hypoxia and cobalt chloride (CoCl2 ), a well-known hypoxia-mimetic agent, have also been reported to induce neurite outgrowth in PC12 cells [10,24,28]. Visfatin, a highly conserved 52-kDa protein, is a novel adipokine that is produced in large amounts in visceral adipose tissue [15].
∗ Corresponding author at: Department of Dental Pharmacology, School of Dentistry, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea. Tel.: +82 51 510 8253, fax: +82 51 510 8233. E-mail address: [email protected] (S.-K. Bae). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.09.014
Visfatin was first reported as a nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis [9], and later rediscovered as a pre-B cell colony-enhancing factor (PBEF) that enhances the effects of interleukin-7 and stem cell factor on pre-B cell colony formation [29]. In addition to these activities, we and others have recently demonstrated that visfatin has angiogenic and inflammatory activities in in vitro, ex vivo, and in vivo assays [1,3,4,20–22,27]. Visfatin has also been implicated in the tumorigenesis and/or metastasis of various cancers [2,5]. Although there is mounting evidence for the role of visfatin in vascular, immune, and cancer cells, very little is known with regard to its biological effects on neuronal cells. Here, we investigate whether visfatin plays a role in neurite outgrowth in PC12 cells and characterize the mechanism involved. U0126 and K252a were obtained from Calbiochem. FK866, an inhibitor of Nampt was from Cayman Chemical. Antirabbit IgG-HRP and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-ERK1/2, mouse monoclonal anti-pERK1/2, and mouse monoclonal anti--actin antibodies were bought from Cell Signaling Technology Inc.
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Fig. 1. Neurite outgrowth is induced by CoCl2 in PC12 cells. (A) PC12 cells were treated with different concentrations of CoCl2 for 24 h and cell viability was measured by CCK-8 assay. n = 3, *p < 0.01; **p < 0.001 vs. control. (B) Cells were treated with 75 M CoCl2 for 24 h and subjected to immunocytochemical analysis of Tuj-1. Cells were stained for Tuj-1 (red) and nuclei were revealed with DAPI (blue). Images were taken at 200× magnification. (C) PC12 cells were incubated with or without 75 M CoCl2 for 24 h in the presence or absence of the indicated inhibitors. PC12 cells were pretreated with or without 100 pM FK866, 10 nM K252a, or 5 nM U0126 for 2 h. Images were taken at 200× magnification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
The rat visfatin ELISA KIT, recombinant human visfatin protein, and mouse monoclonal pan-visfatin antibody were obtained from Adipogen. Mouse monoclonal anti-Tuj-1 was obtained from Millipore. PC12 cells were cultured at 37 ◦ C in growth medium (DMEM/high glucose supplemented with 2 mM l-glutamine, 10% fetal bovine serum (FBS; GIBCO), 5% horse serum (HS; Hyclone), and 1% antibiotics (10,000 units/mL Penicillin and 10,000 g/mL Streptomycin; Hyclone)) in a humidified atmosphere containing 95% air and 5% CO2 . The cells were passaged 2–3 times a week. For the study of neurite outgrowth, PC12 cells were cultured in 0.5% FBS medium
(DMEM/high glucose containing 0.5% FBS, 2 mM l-glutamine, and antibiotics) with CoCl2 or visfatin, for 24 h and 48 h, respectively. Cell viability after treatment with CoCl2 was measured by the Cell Counting Kit-8 (CCK-8) assay. PC12 cells were suspended at a final concentration of 5 × 104 cells/well and cultured in a 24well flat-bottomed microplate. After different treatment of cells, CCK-8 (30 L) was added to each well containing 300 L of the culture medium, and the plate was incubated for 1 h at 37 ◦ C. Viable cells were counted by absorbance measurements at 450 nm using an ELISA reader (Dynex Technologies). All experiments were performed in triplicate on three separate occasions.
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Fig. 2. Visfatin is upregulated by CoCl2 in PC12 cells. (A) Visfatin protein levels were examined by Western blot assay with antibodies against visfatin. -Actin served as the loading control. (B) Quantitative analysis of visfatin protein expression normalized to -actin expression is shown in the right panel. n = 3, *p < 0.001 vs. control. (C) PC12 cells were incubated with 75 M CoCl2 for the indicated times. The amount of visfatin protein secreted into the culture medium was measured by ELISA. n = 3, *p < 0.001 vs. control. (D) Immunocytochemical analysis of visfatin expression. Cells were treated with 75 M CoCl2 for 24 h and subjected to immunocytochemical analysis of visfatin. Cells were stained with visfatin (green) and nuclei were stained with DAPI (blue). Images were taken at 200× magnification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
For Western blot analsysis, harvested cells were lysed in a lysis buffer (40 mM Tris–Cl, 10 mM EDTA, 120 mM NaCl and 0.1% NP-40) containing a protease inhibitor cocktail (Sigma). A constant protein concentration (30 g/lane) was used. The proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 for 1 h at room temperature, and probed with the appropriate antibodies. The signal was developed using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). For analyzing neurite outgrowth, cells were seeded at a density of 5 × 105 cells per well, with each well containing 500 L of culture medium, on 24-well plates. After treatment with CoCl2 or visfatin, neurite outgrowth was photographed under a phase contrast microscope.
For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 5 min, blocked with 5% normal goat serum/PBS for 1 h, labeled with the appropriate primary antibodies and FITCconjugated secondary antibody, and finally treated with DAPI (Vector Laboratories). Cells were analyzed using fluorescence microscopy (Nikon). For enzyme-linked immunosorbent assays (ELISA), PC12 cells were treated with CoCl2 for 48 h. At the end of the treatment, the culture media were collected and centrifuged at 14,000 rpm for 5 min. The supernatants were stored in aliquots at −70 ◦ C. Visfatin levels in the media were determined by ELISA using the human Visfatin ELISA MaxTM Set Deluxe Kit (Adipogen). To investigate the effect of CoCl2 on the viability of PC12 cells, the cells were treated with different concentrations of CoCl2 in 0.5% FBS medium for 24 h, following which the CCK-8 assay was performed. As shown in Fig. 1A, no statistically significant decrease
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Fig. 3. Visfatin induces neurite outgrowth, which is suppressed by ERK1/2 inhibitor U0126. (A) PC12 cells were pre-incubated with U0126 (5 M) for 2 h before exposure to visfatin (100 ng/mL) for 48 h. Images were taken at 200× magnification. (B) The levels of total ERK1/2 and phosphorylated ERK1/2 protein were determined by Western blot analysis. PC12 cells were stimulated with 100 ng/mL visfatin for the indicated times. Phosphorylated forms of ERK1/2 in whole cell extracts were detected using p-ERK1/2-specific antibody. (C) Quantitative analysis of p-ERK1/2 normalized to total ERK1/2 expression is shown in the right panel. n = 3, *p < 0.001 vs. control.
in cell viability was observed for cells treated with 150 M CoCl2 or less. However, at concentrations higher than 200 M, CoCl2 treatment showed a significant decrease in the viability of PC12 cells in a dose-dependent manner. Since both 75 M and 150 M CoCl2 had neuritogenic effects on PC12 cells, we chose lower dose of CoCl2 for the following studies. As shown in Fig. 1B, CoCl2 treated PC12 cells, unlike untreated control cells, expressed typical branched structures and exhibited the projections that characterize neurite outgrowth. When PC12 cells were immunostained with antibody against Tuj-1 (type III -tubulin), a marker for immature neurons [7], the number of CoCl2 -treated cells recognized by the antibody was significantly higher than that of control untreated cells (Fig. 1B). To determine whether CoCl2 -induced neurite outgrowth correlates with induction of visfatin gene expression in PC12 cells, cells were incubated with 75 M CoCl2 , and the levels of visfatin mRNA
were analyzed by RT-PCR. The treatment of CoCl2 resulted in a significant (∼3.2-fold) and time-dependent increase in the levels of visfatin mRNA, compared to the untreated control (Supplementary Fig. 1A and B). The levels of visfatin protein in CoCl2 -treated PC12 cells were further analyzed by Western blot analysis and ELISA. Consistent with the RT-PCR findings, visfatin protein levels in cell lysates increased in a time-dependent manner following CoCl2 treatment (Fig. 2A and B). The release of visfatin protein into the culture medium was also enhanced by CoCl2 treatment (Fig. 2C). The increase in visfatin protein levels was further confirmed by immunocytochemistry. As shown in Fig. 2D, the visfatin signal was markedly higher in CoCl2 -treated cells than in control cells. Visfatin is known to function as a nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the synthesis of NAD+ from nicotinamide [9,16]. To determine whether the Nampt enzymatic activity of visfatin is important for CoCl2 -induced
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neurite outgrowth, PC12 cells were preincubated with FK866, a potent selective Nampt inhibitor [12], followed by 75 M CoCl2 treatment, and then neurite outgrowth was examined. As shown in Fig. 1C, the inhibition of Nampt enzyme activity by FK866 did not affect CoCl2 -induced neurite outgrowth in PC12 cells. Signaling via the NGF–NGF receptor TrkA pathway has been reported to play an important role in neuritogenesis in neuronal cells, including PC12 cells [13,14,32]. Thus, to elucidate whether NGF/TrkA signaling is involved in the induction of neurite outgrowth in PC12 cells by CoCl2 , cells were incubated with 75 M CoCl2 in 0.5% FBS medium for 24 h, and the levels of NGF mRNA were analyzed by RT-PCR. As shown in Supplementary Fig. 1A, NGF did not show any change in mRNA levels after CoCl2 treatment. In addition, PC12 cells were preincubated with K252a, a Trk inhibitor [31], followed by CoCl2 treatment, and then neurite outgrowth was examined. As shown in Fig. 1C, K252a had no effect on CoCl2 -induced neurite outgrowth in PC12 cells. To examine the physiological effect of visfatin within PC12 cells, we examined morphological changes in the cells upon treatment with visfatin. As shown in Fig. 3A, neurite outgrowth was seen in PC12 cells treated with visfatin for 48 h, in contrast to untreated control cells. We next aimed to describe the signaling mechanism through which visfatin contributes to neurite outgrowth. Visfatin has been known to activate endothelial ERK1/2 signaling [3,21], and ERK1/2 activation is an important cellular signaling event for neuritogenesis of PC12 cells [33]; we therefore hypothesized that ERK1/2 signaling may play a role in visfatin-induced neuritogenesis in PC12. In order to test this hypothesis, PC12 cells were exposed to visfatin, and ERK1/2 activation was analyzed by Western blot analysis using antibodies directed against the phosphorylated forms of ERK1/2 (p44 ERK1 and p42 ERK2). As shown in Fig. 3B, visfatin induced ERK1/2 activation in a time-dependent manner; phosphorylation reached maximal levels at 60 min, and persisted for 2 h. To determine the role of ERK1/2 activity on the induction of neuritogenesis by visfatin, PC12 cells were pretreated with U0126, a specific ERK1/2 inhibitor [19], prior to exposure to visfatin and morphological changes were examined. As shown in Fig. 3A, the pretreatment of cells with U0126 blocked visfatin-induced neurite outgrowth in PC12 cells. In addition, to examine whether ERK1/2 activity is involved in CoCl2 -induced neurite outgrowth, PC12 cells were preincubated with U0126 followed by CoCl2 treatment. As shown in Fig. 1C, U0126 had inhibitory effects on CoCl2 -induced neurite outgrowth in PC12 cells. Adipokines such as leptin, angiopoietin-1, NGF, VEGF, IL-6, and FGF-2 have been reported to act as neurotropic factors in promoting neuritogenesis as well as angiogenesis [6,8,17,18,23,30]. We have recently demonstrated that a new adipokine, visfatin, whose expression is upregulated by hypoxia-inducible factor-1 under hypoxic conditions such as 1% O2 or treatment with the hypoxia-mimetic chemical agent CoCl2 , acts as a proangiogenic factor by inducing FGF-2 and IL-6 production in human endothelial cells [2–4,20]. Here, we expanded the study of visfatin to examine its role in PC12 cells, which are widely used as a model for neurite outgrowth. The present study demonstrates for the first time that visfatin is upregulated in the process of CoCl2 -induced neurite outgrowth in PC12 cells, and that visfatin possesses the ability to induce neuritogenesis in PC12 cells, suggesting that visfatin can be considered as a new neurotropic factor like NGF. The role of visfatin/PBEF/Nampt in the brain has been recently reported in a mouse model of cerebral ischemia [34]. In addition, plasma levels of visfatin were found to be elevated in patients with ischemic stroke [26]. While these studies have described a neuronal protective role of visfatin within the nervous system, our current data suggest the role of visfatin in the process of neuronal differentiation including neurite outgrowth in neuronal cells.
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Recently, we have demonstrated that visfatin activates endothelial Notch1 signaling, leading to FGF2 induction and angiogenesis [4]. In PC12 cells, Notch signaling is reported to inhibit neurite outgrowth [25], while FGF2 acts as a positive regulator for neurite outgrowth [17], suggesting that visfatin may exert its effects on PC12 cells via FGF2 upregulation. In addition, IL-6, a factor involved in the induction of neuritogenesis [5], is also upregulated by visfatin in endothelial and immune cells. In this aspect, it will be interesting to study whether visfatin could exert neuritogenic effects on the nervous system via FGF2, IL-6, or other new factors. This possibility is currently under investigation. In conclusion, our findings provide the first evidence of induction of neurite outgrowth in PC12 cells by a newly characterized adipokine, visfatin. Furthermore, we have demonstrated that ERK1/2 signaling contributes, at least in part, to visfatin-induced neuritogenesis. These results provide important clues toward the understanding of the role of visfatin in the nervous system. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0066585) (to S-K. B.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2011.09.014. References [1] R. Adya, B.K. Tan, A. Punn, J. Chen, H.S. Randeva, Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis, Cardiovasc. Res. 78 (2008) 356–365. [2] S.K. Bae, S.R. Kim, J.G. Kim, J.Y. Kim, T.H. Koo, H.O. Jang, I. Yun, M.A. Yoo, M.K. Bae, Hpoxic induction of human visfatin gene is directly mediated by hypoxiainducible factor-1, FEBS Lett. 580 (2006) 4105–4113. [3] Y.H. Bae, M.K. Bae, S.R. Kim, J.H. Lee, H.J. Wee, S.K. Bae, Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis, Biochem. Biophys. Res. Commun. 379 (2009) 206–211. [4] Y.H. Bae, H.J. Park, S.R. Kim, J.Y. Kim, Y. Kang, J.A. Kim, H.J. Wee, R. Kageyama, J.S. Jung, M.K. Bae, S.K. Bae, Notch1 mediates visfatin-induced FGF-2 up-regulation and endothelial angiogenesis, Cardiovasc. Res. 89 (2011) 436–445. [5] T.Q. Bi, X.M. Che, Nampt/PBEF/visfatin and cancer, Cancer Biol. Ther. 10 (2010) 119–125. [6] S.G. Bouret, S.J. Draper, R.B. Simerly, Trophic action of leptin on hypothalamic neurons that regulate feeding, Science 304 (2004) 108–110. [7] D. Caccamo, C.D. Katsetos, M.M. Herman, A. Frankfurter, V.P. Collins, L.J. Rubinstein, Immunohistochemistry of a spontaneous murine ovarian teratoma with neuroepithelial differentiation. Neuron-associated beta-tubulin as a marker for primitive neuroepithelium, Lab. Invest. 60 (1989) 390–398. [8] E.J. Calabrese, Enhancing and regulating neurite outgrowth, Crit. Rev. Toxicol. 38 (2008) 391–418. [9] L.S. Dietrich, L. Fuller, I.L. Yero, L. Martinez, Nicotinamide mononucleotide pyrophosphorylase activity in animal tissues, J. Biol. Chem. 241 (1966) 188–191. [10] D.C. Genetos, W.K. Cheung, M.L. Decaris, J.K. Leach, Oxygen tension modulates neurite outgrowth in PC12 cells through a mechanism involving HIF and VEGF, J. Mol. Neurosci. 40 (2010) 360–366. [11] 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. [12] M. Hasmann, I. Schemainda, FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis, Cancer Res. 63 (2003) 7436–7442. [13] E.J. Huang, L.F. Reichardt, Neurotrophins: roles in neuronal development and function, Annu. Rev. Neurosci. 24 (2001) 677–736. [14] E.J. Huang, L.F. Reichardt, Trk receptors: roles in neuronal signal transduction, Annu. Rev. Biochem. 72 (2003) 609–642. [15] C. Hug, H.F. Lodish, Medicine. Visfatin: a new adipokine, Science 307 (2005) 366–367. [16] S. Imai, Nicotinamide phosphoribosyltransferase (Nampt): a link between NAD biology, metabolism, and diseases, Curr. Pharm. Des. 15 (2009) 20–28.
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Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Visfatin induces neurite outgrowth in PC12 cells via ERK1/2 signaling pathway Young-Soon Kang a , Moon-Kyoung Bae b,e , Jee-Young Kim c , Joo-Won Jeong d , Il Yun a,e , Hye-Ock Jang a,e , Soo-Kyung Bae a,e,f,∗ a
Department of Dental Pharmacology, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea Department of Oral Physiology, School of Dentistry, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea Department of Physiology, School of Medicine, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea d Department of Anatomy and Neurobiology, School of Medicine, Kyunghee University, Seoul 130-701, South Korea e Research Institute for Oral Biotechnology, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea f Medical Research Institute, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea b c
a r t i c l e
i n f o
Article history: Received 21 July 2011 Received in revised form 25 August 2011 Accepted 11 September 2011 Keywords: CoCl2 ERK1/2 Neurite outgrowth PC12 cells Visfatin
a b s t r a c t The angiogenic and inflammatory functions of visfatin and its effect on vascular cells, are fairly well known. However, its role within the nervous system remains largely unclear. To gain insight into this area, we studied the neuritogenic effect of visfatin on PC12 rat pheochromocytoma cells. We investigated whether visfatin gene expression, which is upregulated by hypoxia in cancer cells, is associated with neuritogenesis in PC12 cells. Using RT-PCR, Western blot analysis, ELISA, morphological observations, and immunostaining, we initially showed that CoCl2 , a hypoxic mimetic agent, upregulated visfatin gene expression along with neurite outgrowth in PC12 cells. We also showed that visfatin stimulated neurite outgrowth in PC12 cells. Moreover, in PC12 cells, visfatin evoked the activation of the extracellular signalregulated kinase 1/2 (ERK1/2), which is closely linked to neuritogenesis. Visfatin-induced outgrowth of neurites was prevented by inhibition of the ERK1/2 pathway. Taken together, our results demonstrate for the first time that visfatin induces neurite outgrowth in PC12 cells via the activation of an ERK-dependent pathway, and suggest that visfatin may exert various biological, physiological, and pathological functions in not only the vascular system but also the nervous system. © 2011 Elsevier Ireland Ltd. All rights reserved.
Precise regulation of neurite outgrowth is critical for axonal growth in neurogenesis and provides the fundamental structure involved in neuronal transmission [8]. PC12 cells, derived from a rat pheochromocytoma, are a well-established model for studying neurite outgrowth; the first report on PC12 cells described neurite outgrowth by nerve growth factor (NGF) [11]. PC12 cells treated with neurotrophic factors, such as NGF, brain derived neurotrophic factor (BDNF), and fibroblast growth factor (FGF), cease division, extend neurites, and differentiate [8,13,32]. This process is largely mediated through TrkA, the high-affinity NGF receptor, leading to the activation of the extracellular signal-related kinase 1/2 (ERK1/2) pathway [14]. Hypoxia and cobalt chloride (CoCl2 ), a well-known hypoxia-mimetic agent, have also been reported to induce neurite outgrowth in PC12 cells [10,24,28]. Visfatin, a highly conserved 52-kDa protein, is a novel adipokine that is produced in large amounts in visceral adipose tissue [15].
∗ Corresponding author at: Department of Dental Pharmacology, School of Dentistry, Yangsan Campus of Pusan National University, Yangsan 626-870, South Korea. Tel.: +82 51 510 8253, fax: +82 51 510 8233. E-mail address: [email protected] (S.-K. Bae). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.09.014
Visfatin was first reported as a nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis [9], and later rediscovered as a pre-B cell colony-enhancing factor (PBEF) that enhances the effects of interleukin-7 and stem cell factor on pre-B cell colony formation [29]. In addition to these activities, we and others have recently demonstrated that visfatin has angiogenic and inflammatory activities in in vitro, ex vivo, and in vivo assays [1,3,4,20–22,27]. Visfatin has also been implicated in the tumorigenesis and/or metastasis of various cancers [2,5]. Although there is mounting evidence for the role of visfatin in vascular, immune, and cancer cells, very little is known with regard to its biological effects on neuronal cells. Here, we investigate whether visfatin plays a role in neurite outgrowth in PC12 cells and characterize the mechanism involved. U0126 and K252a were obtained from Calbiochem. FK866, an inhibitor of Nampt was from Cayman Chemical. Antirabbit IgG-HRP and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-ERK1/2, mouse monoclonal anti-pERK1/2, and mouse monoclonal anti--actin antibodies were bought from Cell Signaling Technology Inc.
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Fig. 1. Neurite outgrowth is induced by CoCl2 in PC12 cells. (A) PC12 cells were treated with different concentrations of CoCl2 for 24 h and cell viability was measured by CCK-8 assay. n = 3, *p < 0.01; **p < 0.001 vs. control. (B) Cells were treated with 75 M CoCl2 for 24 h and subjected to immunocytochemical analysis of Tuj-1. Cells were stained for Tuj-1 (red) and nuclei were revealed with DAPI (blue). Images were taken at 200× magnification. (C) PC12 cells were incubated with or without 75 M CoCl2 for 24 h in the presence or absence of the indicated inhibitors. PC12 cells were pretreated with or without 100 pM FK866, 10 nM K252a, or 5 nM U0126 for 2 h. Images were taken at 200× magnification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
The rat visfatin ELISA KIT, recombinant human visfatin protein, and mouse monoclonal pan-visfatin antibody were obtained from Adipogen. Mouse monoclonal anti-Tuj-1 was obtained from Millipore. PC12 cells were cultured at 37 ◦ C in growth medium (DMEM/high glucose supplemented with 2 mM l-glutamine, 10% fetal bovine serum (FBS; GIBCO), 5% horse serum (HS; Hyclone), and 1% antibiotics (10,000 units/mL Penicillin and 10,000 g/mL Streptomycin; Hyclone)) in a humidified atmosphere containing 95% air and 5% CO2 . The cells were passaged 2–3 times a week. For the study of neurite outgrowth, PC12 cells were cultured in 0.5% FBS medium
(DMEM/high glucose containing 0.5% FBS, 2 mM l-glutamine, and antibiotics) with CoCl2 or visfatin, for 24 h and 48 h, respectively. Cell viability after treatment with CoCl2 was measured by the Cell Counting Kit-8 (CCK-8) assay. PC12 cells were suspended at a final concentration of 5 × 104 cells/well and cultured in a 24well flat-bottomed microplate. After different treatment of cells, CCK-8 (30 L) was added to each well containing 300 L of the culture medium, and the plate was incubated for 1 h at 37 ◦ C. Viable cells were counted by absorbance measurements at 450 nm using an ELISA reader (Dynex Technologies). All experiments were performed in triplicate on three separate occasions.
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Fig. 2. Visfatin is upregulated by CoCl2 in PC12 cells. (A) Visfatin protein levels were examined by Western blot assay with antibodies against visfatin. -Actin served as the loading control. (B) Quantitative analysis of visfatin protein expression normalized to -actin expression is shown in the right panel. n = 3, *p < 0.001 vs. control. (C) PC12 cells were incubated with 75 M CoCl2 for the indicated times. The amount of visfatin protein secreted into the culture medium was measured by ELISA. n = 3, *p < 0.001 vs. control. (D) Immunocytochemical analysis of visfatin expression. Cells were treated with 75 M CoCl2 for 24 h and subjected to immunocytochemical analysis of visfatin. Cells were stained with visfatin (green) and nuclei were stained with DAPI (blue). Images were taken at 200× magnification. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
For Western blot analsysis, harvested cells were lysed in a lysis buffer (40 mM Tris–Cl, 10 mM EDTA, 120 mM NaCl and 0.1% NP-40) containing a protease inhibitor cocktail (Sigma). A constant protein concentration (30 g/lane) was used. The proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 for 1 h at room temperature, and probed with the appropriate antibodies. The signal was developed using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). For analyzing neurite outgrowth, cells were seeded at a density of 5 × 105 cells per well, with each well containing 500 L of culture medium, on 24-well plates. After treatment with CoCl2 or visfatin, neurite outgrowth was photographed under a phase contrast microscope.
For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 5 min, blocked with 5% normal goat serum/PBS for 1 h, labeled with the appropriate primary antibodies and FITCconjugated secondary antibody, and finally treated with DAPI (Vector Laboratories). Cells were analyzed using fluorescence microscopy (Nikon). For enzyme-linked immunosorbent assays (ELISA), PC12 cells were treated with CoCl2 for 48 h. At the end of the treatment, the culture media were collected and centrifuged at 14,000 rpm for 5 min. The supernatants were stored in aliquots at −70 ◦ C. Visfatin levels in the media were determined by ELISA using the human Visfatin ELISA MaxTM Set Deluxe Kit (Adipogen). To investigate the effect of CoCl2 on the viability of PC12 cells, the cells were treated with different concentrations of CoCl2 in 0.5% FBS medium for 24 h, following which the CCK-8 assay was performed. As shown in Fig. 1A, no statistically significant decrease
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Fig. 3. Visfatin induces neurite outgrowth, which is suppressed by ERK1/2 inhibitor U0126. (A) PC12 cells were pre-incubated with U0126 (5 M) for 2 h before exposure to visfatin (100 ng/mL) for 48 h. Images were taken at 200× magnification. (B) The levels of total ERK1/2 and phosphorylated ERK1/2 protein were determined by Western blot analysis. PC12 cells were stimulated with 100 ng/mL visfatin for the indicated times. Phosphorylated forms of ERK1/2 in whole cell extracts were detected using p-ERK1/2-specific antibody. (C) Quantitative analysis of p-ERK1/2 normalized to total ERK1/2 expression is shown in the right panel. n = 3, *p < 0.001 vs. control.
in cell viability was observed for cells treated with 150 M CoCl2 or less. However, at concentrations higher than 200 M, CoCl2 treatment showed a significant decrease in the viability of PC12 cells in a dose-dependent manner. Since both 75 M and 150 M CoCl2 had neuritogenic effects on PC12 cells, we chose lower dose of CoCl2 for the following studies. As shown in Fig. 1B, CoCl2 treated PC12 cells, unlike untreated control cells, expressed typical branched structures and exhibited the projections that characterize neurite outgrowth. When PC12 cells were immunostained with antibody against Tuj-1 (type III -tubulin), a marker for immature neurons [7], the number of CoCl2 -treated cells recognized by the antibody was significantly higher than that of control untreated cells (Fig. 1B). To determine whether CoCl2 -induced neurite outgrowth correlates with induction of visfatin gene expression in PC12 cells, cells were incubated with 75 M CoCl2 , and the levels of visfatin mRNA
were analyzed by RT-PCR. The treatment of CoCl2 resulted in a significant (∼3.2-fold) and time-dependent increase in the levels of visfatin mRNA, compared to the untreated control (Supplementary Fig. 1A and B). The levels of visfatin protein in CoCl2 -treated PC12 cells were further analyzed by Western blot analysis and ELISA. Consistent with the RT-PCR findings, visfatin protein levels in cell lysates increased in a time-dependent manner following CoCl2 treatment (Fig. 2A and B). The release of visfatin protein into the culture medium was also enhanced by CoCl2 treatment (Fig. 2C). The increase in visfatin protein levels was further confirmed by immunocytochemistry. As shown in Fig. 2D, the visfatin signal was markedly higher in CoCl2 -treated cells than in control cells. Visfatin is known to function as a nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the synthesis of NAD+ from nicotinamide [9,16]. To determine whether the Nampt enzymatic activity of visfatin is important for CoCl2 -induced
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neurite outgrowth, PC12 cells were preincubated with FK866, a potent selective Nampt inhibitor [12], followed by 75 M CoCl2 treatment, and then neurite outgrowth was examined. As shown in Fig. 1C, the inhibition of Nampt enzyme activity by FK866 did not affect CoCl2 -induced neurite outgrowth in PC12 cells. Signaling via the NGF–NGF receptor TrkA pathway has been reported to play an important role in neuritogenesis in neuronal cells, including PC12 cells [13,14,32]. Thus, to elucidate whether NGF/TrkA signaling is involved in the induction of neurite outgrowth in PC12 cells by CoCl2 , cells were incubated with 75 M CoCl2 in 0.5% FBS medium for 24 h, and the levels of NGF mRNA were analyzed by RT-PCR. As shown in Supplementary Fig. 1A, NGF did not show any change in mRNA levels after CoCl2 treatment. In addition, PC12 cells were preincubated with K252a, a Trk inhibitor [31], followed by CoCl2 treatment, and then neurite outgrowth was examined. As shown in Fig. 1C, K252a had no effect on CoCl2 -induced neurite outgrowth in PC12 cells. To examine the physiological effect of visfatin within PC12 cells, we examined morphological changes in the cells upon treatment with visfatin. As shown in Fig. 3A, neurite outgrowth was seen in PC12 cells treated with visfatin for 48 h, in contrast to untreated control cells. We next aimed to describe the signaling mechanism through which visfatin contributes to neurite outgrowth. Visfatin has been known to activate endothelial ERK1/2 signaling [3,21], and ERK1/2 activation is an important cellular signaling event for neuritogenesis of PC12 cells [33]; we therefore hypothesized that ERK1/2 signaling may play a role in visfatin-induced neuritogenesis in PC12. In order to test this hypothesis, PC12 cells were exposed to visfatin, and ERK1/2 activation was analyzed by Western blot analysis using antibodies directed against the phosphorylated forms of ERK1/2 (p44 ERK1 and p42 ERK2). As shown in Fig. 3B, visfatin induced ERK1/2 activation in a time-dependent manner; phosphorylation reached maximal levels at 60 min, and persisted for 2 h. To determine the role of ERK1/2 activity on the induction of neuritogenesis by visfatin, PC12 cells were pretreated with U0126, a specific ERK1/2 inhibitor [19], prior to exposure to visfatin and morphological changes were examined. As shown in Fig. 3A, the pretreatment of cells with U0126 blocked visfatin-induced neurite outgrowth in PC12 cells. In addition, to examine whether ERK1/2 activity is involved in CoCl2 -induced neurite outgrowth, PC12 cells were preincubated with U0126 followed by CoCl2 treatment. As shown in Fig. 1C, U0126 had inhibitory effects on CoCl2 -induced neurite outgrowth in PC12 cells. Adipokines such as leptin, angiopoietin-1, NGF, VEGF, IL-6, and FGF-2 have been reported to act as neurotropic factors in promoting neuritogenesis as well as angiogenesis [6,8,17,18,23,30]. We have recently demonstrated that a new adipokine, visfatin, whose expression is upregulated by hypoxia-inducible factor-1 under hypoxic conditions such as 1% O2 or treatment with the hypoxia-mimetic chemical agent CoCl2 , acts as a proangiogenic factor by inducing FGF-2 and IL-6 production in human endothelial cells [2–4,20]. Here, we expanded the study of visfatin to examine its role in PC12 cells, which are widely used as a model for neurite outgrowth. The present study demonstrates for the first time that visfatin is upregulated in the process of CoCl2 -induced neurite outgrowth in PC12 cells, and that visfatin possesses the ability to induce neuritogenesis in PC12 cells, suggesting that visfatin can be considered as a new neurotropic factor like NGF. The role of visfatin/PBEF/Nampt in the brain has been recently reported in a mouse model of cerebral ischemia [34]. In addition, plasma levels of visfatin were found to be elevated in patients with ischemic stroke [26]. While these studies have described a neuronal protective role of visfatin within the nervous system, our current data suggest the role of visfatin in the process of neuronal differentiation including neurite outgrowth in neuronal cells.
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Recently, we have demonstrated that visfatin activates endothelial Notch1 signaling, leading to FGF2 induction and angiogenesis [4]. In PC12 cells, Notch signaling is reported to inhibit neurite outgrowth [25], while FGF2 acts as a positive regulator for neurite outgrowth [17], suggesting that visfatin may exert its effects on PC12 cells via FGF2 upregulation. In addition, IL-6, a factor involved in the induction of neuritogenesis [5], is also upregulated by visfatin in endothelial and immune cells. In this aspect, it will be interesting to study whether visfatin could exert neuritogenic effects on the nervous system via FGF2, IL-6, or other new factors. This possibility is currently under investigation. In conclusion, our findings provide the first evidence of induction of neurite outgrowth in PC12 cells by a newly characterized adipokine, visfatin. Furthermore, we have demonstrated that ERK1/2 signaling contributes, at least in part, to visfatin-induced neuritogenesis. These results provide important clues toward the understanding of the role of visfatin in the nervous system. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0066585) (to S-K. B.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2011.09.014. References [1] R. Adya, B.K. Tan, A. Punn, J. Chen, H.S. Randeva, Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis, Cardiovasc. Res. 78 (2008) 356–365. [2] S.K. Bae, S.R. Kim, J.G. Kim, J.Y. Kim, T.H. Koo, H.O. Jang, I. Yun, M.A. Yoo, M.K. Bae, Hpoxic induction of human visfatin gene is directly mediated by hypoxiainducible factor-1, FEBS Lett. 580 (2006) 4105–4113. [3] Y.H. Bae, M.K. Bae, S.R. Kim, J.H. Lee, H.J. Wee, S.K. Bae, Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis, Biochem. Biophys. Res. Commun. 379 (2009) 206–211. [4] Y.H. Bae, H.J. Park, S.R. Kim, J.Y. Kim, Y. Kang, J.A. Kim, H.J. Wee, R. Kageyama, J.S. Jung, M.K. Bae, S.K. Bae, Notch1 mediates visfatin-induced FGF-2 up-regulation and endothelial angiogenesis, Cardiovasc. Res. 89 (2011) 436–445. [5] T.Q. Bi, X.M. Che, Nampt/PBEF/visfatin and cancer, Cancer Biol. Ther. 10 (2010) 119–125. [6] S.G. Bouret, S.J. Draper, R.B. Simerly, Trophic action of leptin on hypothalamic neurons that regulate feeding, Science 304 (2004) 108–110. [7] D. Caccamo, C.D. Katsetos, M.M. Herman, A. Frankfurter, V.P. Collins, L.J. Rubinstein, Immunohistochemistry of a spontaneous murine ovarian teratoma with neuroepithelial differentiation. Neuron-associated beta-tubulin as a marker for primitive neuroepithelium, Lab. Invest. 60 (1989) 390–398. [8] E.J. Calabrese, Enhancing and regulating neurite outgrowth, Crit. Rev. Toxicol. 38 (2008) 391–418. [9] L.S. Dietrich, L. Fuller, I.L. Yero, L. Martinez, Nicotinamide mononucleotide pyrophosphorylase activity in animal tissues, J. Biol. Chem. 241 (1966) 188–191. [10] D.C. Genetos, W.K. Cheung, M.L. Decaris, J.K. Leach, Oxygen tension modulates neurite outgrowth in PC12 cells through a mechanism involving HIF and VEGF, J. Mol. Neurosci. 40 (2010) 360–366. [11] 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. [12] M. Hasmann, I. Schemainda, FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis, Cancer Res. 63 (2003) 7436–7442. [13] E.J. Huang, L.F. Reichardt, Neurotrophins: roles in neuronal development and function, Annu. Rev. Neurosci. 24 (2001) 677–736. [14] E.J. Huang, L.F. Reichardt, Trk receptors: roles in neuronal signal transduction, Annu. Rev. Biochem. 72 (2003) 609–642. [15] C. Hug, H.F. Lodish, Medicine. Visfatin: a new adipokine, Science 307 (2005) 366–367. [16] S. Imai, Nicotinamide phosphoribosyltransferase (Nampt): a link between NAD biology, metabolism, and diseases, Curr. Pharm. Des. 15 (2009) 20–28.
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