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NO is not involved in the simvastatin induced cell division and differentiation in PC12 cells
Neuroscience Letters 243 (1998) 73–76
NO is not involved in the simvastatin induced cell division and differentiation in PC12 cells Masahiro Sano a , b, Ikuko Sato-Suzuki a, Hiroshi Fujita a, Ikuo Morita a, Masanori Nagao b, Sei-itsu Murota a ,* a
Department of Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan b Department of Geriatric Dentistry, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan Received 11 September 1997; received in revised form 6 January 1998; accepted 16 January 1998
Abstract Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor has been reported to inhibit cell division and induce neurite-like outgrowth in PC12 cells [Sato-Suzuki, I. and Murota, S., Neurosci. Lett., 220 (1996) 21–24]. In the present paper, we examined whether the induced nitric oxide (NO) in the simvastatin-treated PC12 cells is involved in the growth arrest and differentiation as reported in nerve growth factor (NGF) treated PC12 cells. Treatment of PC12 cells with simvastatin caused peripherin formation and enhanced NO production just like NGF-treated PC12 cells. Different from NGF, however, NO synthase inhibitors could not affect the growth arrest and differentiation in simvastatin-treated PC12 cells. In conclusion, NO had nothing to do with cell division and differentiation in simvastatin-treated PC12 cells. 1998 Elsevier Science Ireland Ltd.
Keywords: Simvastatin; 3-Hydroxy-3-methylglutaryl coenzyme A reductase; PC12; Nitric oxide; Cell division; Neurite outgrowth
Simvastatin is a potent inhibitor of cholesterol synthesis both in vitro and in vivo due to the competitive suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme for cholesterol biosynthesis through mevalonate pathway [6,9]. Recently we demonstrated that simvastatin inhibited cell division and induced neurite-like outgrowth in PC12 cells [14]. During the course of the investigation of the mechanism of lovastatin, one of the similar HMG-CoA reductase inhibitors, it has been demonstrated that lovastatin arrests HeLa cells in G1 phase of their cell cycle by maintaining inactivate cyclin E/Cdk (Cyclin-dependent kinase) 2 and cyclin A/Cdk 2 complexes at high levels [10]. Cdk has been shown to drive cell cycle in eukaryotic organisms ranging from yeast to humans [10]. Therefore, an HMG-CoA reductase may be a key enzyme deeply involved in the mechanisms of cell division and differentiation. Nerve growth factor (NGF) has cytostatic effects, such as inducing neurite and nitric oxide synthase (NOS) in neuro* Corresponding author. Fax: +81 3 58030212.
nal cells. The induced nitric oxide (NO) acts as a cytostatic agent in these cells [11]. Natalia and Grigori [11] insisted that NO was involved in neurite outgrowth in NGF-treated PC12 cells. In light of these studies, we first tried to visualize peripherin which was known to be expressed when neuronal cells were differentiated in response to NGF [3], and we confirmed that the neurite-like outgrowth of PC12 cells treated with simvastatin were real neurites, i.e. similar to those outgrown in response to NGF. Additional data showed that simvastatin also induced NO production in the neuronal cells in a dose-dependent fashion. We examined whether NO produced by simvastatin-treated PC12 cells could cause the arrest of cell division, as a result we clarified that the NO released from the simvastatin-treated PC12 cells had nothing to do with the cell division and differentiation. PC12 (Pheochromocytoma) cells were used as undifferentiated neuronal cells in this study. The PC12 cell line cloned at the National Defense Medical College (Kanagawa, Japan) was used. The cells were inoculated into col-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00086- X
74
M. Sano et al. / Neuroscience Letters 243 (1998) 73–76
lagen-coated wells (16 mm, 200 mm2; Iwaki Glass, Japan) with a Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, NY, USA) supplemented with 10% horse serum (HS; GIBCO, NY, USA) and 5% fetal bovine serum (FBS; Bioserum, Australia) and maintained at 37°C in an incubator under a humidified atmosphere of 95% air and 5% CO2. Cells were fixed with 4% paraformaldehyde (Sigma, MO, USA) for 20 min at room temperature after the incubation with simvastatin (1 mg/ml; Merk, Japan) for 24 h or NGF (50 ng/ml, Biomedical Technologies, MA, USA) for 72 h. We also prepared the same conditions with NOS inhibitor, Nnitro-L-arginine methyl ester (L-NAME; Sigma, MO, USA). Then the fixed cells were incubated overnight at 4°C with anti-peripherin monoclonal antibody (Chemicon International, CA, USA), and then they were stained according to the biotin-streptavidin horseradish peroxidase method (Sensi TekTM HRP, Scytek, UT, USA). Cell division was determined by measuring the cell number by a Coulter Counter ZBI (Coulter Electronics, Health, FL, USA). For this measurement, each cell culture was
Fig. 2. NO production in PC12 cells treated with simvastatin (24 h) or NGF (72 h). (a) Cells were treated with simvastatin in a dose-dependent fashion (1, 5 and 10 mg/ml). The cells treated with simvastatin (10 mg/ml) were concomitantly treated with NOS inhibitors (L-NAME, 2 mM; L-NMMA, 2 mM). (b) Cells were treated with NGF (50 ng/ml) with or without 2 mM of L-NAME. The values of NO production in the control cells represents as 100%. The amount of NOx in the control medium was 3.53 ± 0.78 pmol. Data are the mean ± SEM of three to four cultures. *P , 0.05, compared to the control; **P , 0.05, compared to the medium treated with NGF (50 ng/ml); *P , 0.05, compared to the medium treated with simvastatin (10 mg/ml).
Fig. 1. Immunostaining with anti-peripherin antibody in simvastatintreated (1 mg/ml, 24 h) PC12 cells with or without 2 mM of L-NAME and in NGF-treated (50 ng/ml, 72 h) PC12 cells with or without 2 mM of L-NAME. The expression of peripherin is evident in neurites in both cases (b,c,e). (a) Control, (b) NGF, (c) Simvastatin, (d) NGF and LNAME, (e) Simvastatin and L-NAME. Scale bar, 100 mm.
washed with a phosphate-buffered saline. A mixture of 0.5 ml of 0.05% trypsin and 0.05% EDTA was then used to get a cell suspension. The reactions were terminated by adding 0.5 ml of the growth medium to the cell suspension. NO released from PC12 cells was measured as its NOx metabolites (nitrate, nitrite) in the medium, by means of a chemiluminescence detector (Sievers Instruments, CO, USA). A standard curve was constructed using 50–250 pmol NaNO3 for calculation of picomolar NOx content per sample. The background signal in the medium was subtracted from each measured value. NO production was calculated as follows: NO production = test − blank/cell number (105 cells), the blank was the NOx metabolites
M. Sano et al. / Neuroscience Letters 243 (1998) 73–76
(nitrate, nitrite) from DMEM supplemented with serum. NO production in the control culture was expressed as 100%. Data are expressed as the mean ± SEM, and were analyzed by one-way analysis of variance followed by Fisher’s test to correct for multiple comparisons of treatment. Fig. 1 shows the light microscopic observation of the PC12 cells immunostained with anti-peripherin antibody. Simvastatin-treatment caused the cells to become round with long neurites. The neurites outgrown from the cells treated with both simvastatin and NGF were confirmed to contain peripherin protein (Fig. 1b,c). When these cells were concomitantly treated with 2 mM of L-NAME, the cells treated with NGF did not show neurite formation while the cells with simvastatin did (Fig. 1d,e). Fig. 2a shows NO production in the simvastatin-treated PC12 cells for 24 h. Cells were plated at the density of 3 × 105 cells/ml. The cells were treated with simvastatin in a dose-dependent fashion (1, 5, 10 mg/ml). The cells treated with simvastatin at a concentration of 10 mg/ml were also treated with one of NOS inhibitors concomitantly. L-NAME (2 mM) and of N-monomethyl-L-arginine (L-NMMA; 2 mM; Sigma, MO, USA) were used as NOS inhibitors. Simvastatin treatment increased NO production in PC12 cells in a dose-dependent fashion, and NOS inhibitors significantly suppressed the increase. Fig. 2b shows the data on the NGF treatment (50 ng/ml) for 72 h. In this case, cells were plated at the density of 105 cells/ml for the longer period (72 h) of the culture. L-NAME suppressed the increase in NO production in the PC12 cells treated with NGF. The control medium contained 3.53 ± 0.78 pmol of NOx. Fig. 3 shows the effects of simvastatin treatment on the division of PC12 cells. Cells were treated with simvastatin (10 mg/ml) with or without NOS inhibitors (L-NAME, 2 mM; L-NMMA, 2 mM) for 24 h. The growth was remark-
Fig. 3. The effects of simvastatin on the division of PC12 cells. The number of cells was counted 24 h after the treatment with simvastatin (10 mg/ml) with or without NOS inhibitors (L-NAME, 2 mM; LNMMA, 2 mM). Data are the mean ± SEM for three to four cultures. *P , 0.05, compared to the control.
75
ably inhibited by the simvastatin treatment and NOS inhibitors could not rescue it. In the parallel experiment with NGF (50 ng/ml, 72 h) instead of simvastatin, the decrease in the cell number was limited. However, in the case of NGF, L-NAME (2 mM) recovered the decrease in the cell number as previously reported [11] (data not shown). In our previous study, we found that simvastatin induced neurite-like outgrowth in PC12 cells [14], but we did not examine whether the outgrowth was accompanied by peripherin expression. In the present study, the expression of peripherin was clearly demonstrated in the neurite-like outgrowth of PC12 cells treated with simvastatin. Thus we confirmed that simvastatin caused real neurite outgrowth in PC12 cells. Peripherin is a protein which is expressed when neuronal cells differentiate under such condition as NGF treatment [3]. Our data shows that the neurite outgrowth of PC12 cells treated with simvastatin is the same as that shown in NGF treated PC12 cells from the viewpoint of peripherin formation. However, there are several differences in the neurite outgrowth in PC12 cells between simvastatin and general neurotrophic factors such as NGF. Firstly, simvastatin can induce the neurite outgrowth rapidly, i.e. within only 20 h from the start of simvastatin treatment [14], while in the case of NGF, it takes at least 2 days to get neurite-outgrowth in PC12 cells [3]. Secondly, the shape of simvastatin-treated cells is different from that of NGF-treated ones. Simvastatin causes formation of a roundish cell body while NGF treated cells remain polygonal. Simvastatin treated patients are sometimes known to be suffering from central nervous system (CNS) adverse effects [2,4,13,15]. The neuronal cells may be damaged in some way by the simvastatin treatment. In our present study simvastatin inhibited the cell division of PC12 cells. The inhibition of neuronal cell division could be related to the CNS adverse effects. A recent study demonstrates that NO is involved in the cytostatic effect of NGF on PC12 cells [11]. They claimed that both NO and NGF were necessary to be present at the same time to induce differentiation and arrest cell cycle in PC12 cells because in iNOS (−/−) mutant cells NO donor alone or NGF alone failed to induce differentiation and arrest cell cycle in the cells [11]. In this study we showed that NO from NGF-treated PC12 cells was involved in the differentiation of PC12 cells because NOS inhibitor inhibited NGF induced growth arrest and cell differentiation. We first thought that NO from simvastatin-treated PC12 cells was also involved in the differentiation of PC12 cells. However, NO had nothing to do with differentiation in simvastatin-treated PC12 cells, because NOS inhibitors could not abolish simvastatin induced PC12 cell differentiation. So we speculate that the mechanism by which simvastatin induces cytostatic action and differentiation is different from those induced by NGF. Although the mechanisms by which simvastatin stimulates NO production in PC12 cells are still unclear, simvas-
76
M. Sano et al. / Neuroscience Letters 243 (1998) 73–76
tatin may induce NOS. In our preliminary experiments, simvastatin-treated PC12 cells were stained with anti-NOS antibody (data not shown). Furthermore, it has been reported that membrane cholesterol contents affect uptake capacity of calcium ion in cells [1,5]. Although we did not examine whether PC12 cells treated with simvastatin are enhanced in the Ca2+ uptake activity, the Ca2+ may activate Ca2+-dependent NOS activities such as eNOS or nNOS activity. Lovastatin induces cyclin-dependent kinase inhibitors (P21, P27) in human breast cancer cells in a cell cycleindependent fashion [7,8]. Cell cycle phase transitions in eukaryotic cells are driven by the regulation of the activity of protein kinases known as cyclin-dependent kinases (Cdk). p27 is redistributed from cyclin D1/Cdk4 to cyclin A/Cdk2 complexes in Swiss 3T3 mouse fibroblast cells treated with lovastatin [12]. It is likely that a similar principle of redistribution of p27 is seen in other instances of cell cycle arrest. Therefore HMG Co-A reductase may be the key enzyme which correlates to the mechanisms of cell division and differentiation. Although the reason why NO is not involved in the all static action and differentiation is unclear in simvastatin-treated cells, HMG-CoA reductase inhibitor may be a single differentiation factor that is not effected by NO. In conclusion, we demonstrate that simvastatin induces NO production in PC12 cells in a dose-dependent fashion. The neurite outgrown in simvastatin-treated PC12 cells is similar to the one seen in NGF-treated PC12 cells, but the mechanism of NO production, growth arrest and differentiation induced by these two substances are different. Further study is required to make these mechanisms clear. The authors thank Dr. Toshiyuki Himi, Tokyo Medical and Dental University, for helpful suggestions and critical discussion of the manuscript. We also thank Dr. Hitoshi Wakisaka, National Defense Medical College, for kindly providing PC12 cells. [1] Bialecki, RA. and Tulenko, TN., Excess membrane cholesterol alters calcium channels in arterial smooth muscle, Am. J. Physiol., 257 (1989) C306–C314.
[2] Chariot, P., Abadia, R., Danan, C. and Charpentier, C., Simvastatin-induced rhabdomyolysis followed by a melas syndrome, Am. J. Med., 94 (1993) 109–110. [3] Demarais, D. and Royal, A., The TATA motif is a target for efficient transcriptionl activation and nerve growth factor induction of the peripherin gene, J. Biol. Chem., 271 (40) (1996) 24976–24981. [4] Duits, N. and Bos, F.M., Depressive symptoms and cholesterollowering drugs, Lancet, 341 (1993) 114. [5] Gleason, M.M., Medow, Ms. and Tulenko, T.N., Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells, Circ. Res., 69 (1) (1991) 216–227. [6] Goldstein, J.L. and Brown, M.S., Regulation of the mevalonate pathway, Nature, 343 (1990) 425–429. [7] Gray-Bablin, J., Rao, S. and Keyomarsi, K., Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion, Cancer Res., 57 (4 ) (1997) 604–609. [8] Hengst, L. and Reed, S.I., Translational control of p27Kipl accumulation during the cell cycle, Science, 271 (5257) (1996) 1861–1864. [9] Hoffman, W., Albers, A., Anderson, P., Chen, J., Smith, R. and Willard, A., 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 4. Side chain ester derivatives of mevinolin, J. Med. Chem., 29 (1989) 849–852. [10] Ludger, H., Vjekoslav, D., Joyce, M.S., Emma, L. and Steven, I.R., A cell cycle -regulated inhibitor of cyclin-dependent kinases, Cell Biol., 91 (1994) 5291–5295. [11] Natalia, P. and Grigori, E., Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells, Nature, 375 (1995) 68–73. [12] Poon, R.Y., Toyoshima, H. and Hunter, T., Redistribution of the CDK inhibitor p27 between different cyclin. CDK complexes in the mouse fibroblast cell cycle and in the cells arrested with lovastatin or ultraviolet irradiation, Mol. Biol. Cell, 6 (9) (1995) 1197–1213. [13] Saheki, A., Terasaki, T., Tamai, I. and Tsuji, A., In vivo and in vitro blood-brain transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, Pharmacol. Res., 11 (1994) 304–311. [14] Sato-Suzuki, I. and Murota, S., Simvastatin inhibits the division and induces neurite-like outgrowth in PC12 cells, Neurosci. Lett., 220 (1996) 21–24. [15] Smith, G.D. and Pekkanen, J., Should there be a moratorium on the use of cholesterol lowering drugs?, Br. Med. J., 304 (1992) 431–434.
NO is not involved in the simvastatin induced cell division and differentiation in PC12 cells Masahiro Sano a , b, Ikuko Sato-Suzuki a, Hiroshi Fujita a, Ikuo Morita a, Masanori Nagao b, Sei-itsu Murota a ,* a
Department of Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan b Department of Geriatric Dentistry, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan Received 11 September 1997; received in revised form 6 January 1998; accepted 16 January 1998
Abstract Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor has been reported to inhibit cell division and induce neurite-like outgrowth in PC12 cells [Sato-Suzuki, I. and Murota, S., Neurosci. Lett., 220 (1996) 21–24]. In the present paper, we examined whether the induced nitric oxide (NO) in the simvastatin-treated PC12 cells is involved in the growth arrest and differentiation as reported in nerve growth factor (NGF) treated PC12 cells. Treatment of PC12 cells with simvastatin caused peripherin formation and enhanced NO production just like NGF-treated PC12 cells. Different from NGF, however, NO synthase inhibitors could not affect the growth arrest and differentiation in simvastatin-treated PC12 cells. In conclusion, NO had nothing to do with cell division and differentiation in simvastatin-treated PC12 cells. 1998 Elsevier Science Ireland Ltd.
Keywords: Simvastatin; 3-Hydroxy-3-methylglutaryl coenzyme A reductase; PC12; Nitric oxide; Cell division; Neurite outgrowth
Simvastatin is a potent inhibitor of cholesterol synthesis both in vitro and in vivo due to the competitive suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme for cholesterol biosynthesis through mevalonate pathway [6,9]. Recently we demonstrated that simvastatin inhibited cell division and induced neurite-like outgrowth in PC12 cells [14]. During the course of the investigation of the mechanism of lovastatin, one of the similar HMG-CoA reductase inhibitors, it has been demonstrated that lovastatin arrests HeLa cells in G1 phase of their cell cycle by maintaining inactivate cyclin E/Cdk (Cyclin-dependent kinase) 2 and cyclin A/Cdk 2 complexes at high levels [10]. Cdk has been shown to drive cell cycle in eukaryotic organisms ranging from yeast to humans [10]. Therefore, an HMG-CoA reductase may be a key enzyme deeply involved in the mechanisms of cell division and differentiation. Nerve growth factor (NGF) has cytostatic effects, such as inducing neurite and nitric oxide synthase (NOS) in neuro* Corresponding author. Fax: +81 3 58030212.
nal cells. The induced nitric oxide (NO) acts as a cytostatic agent in these cells [11]. Natalia and Grigori [11] insisted that NO was involved in neurite outgrowth in NGF-treated PC12 cells. In light of these studies, we first tried to visualize peripherin which was known to be expressed when neuronal cells were differentiated in response to NGF [3], and we confirmed that the neurite-like outgrowth of PC12 cells treated with simvastatin were real neurites, i.e. similar to those outgrown in response to NGF. Additional data showed that simvastatin also induced NO production in the neuronal cells in a dose-dependent fashion. We examined whether NO produced by simvastatin-treated PC12 cells could cause the arrest of cell division, as a result we clarified that the NO released from the simvastatin-treated PC12 cells had nothing to do with the cell division and differentiation. PC12 (Pheochromocytoma) cells were used as undifferentiated neuronal cells in this study. The PC12 cell line cloned at the National Defense Medical College (Kanagawa, Japan) was used. The cells were inoculated into col-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00086- X
74
M. Sano et al. / Neuroscience Letters 243 (1998) 73–76
lagen-coated wells (16 mm, 200 mm2; Iwaki Glass, Japan) with a Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, NY, USA) supplemented with 10% horse serum (HS; GIBCO, NY, USA) and 5% fetal bovine serum (FBS; Bioserum, Australia) and maintained at 37°C in an incubator under a humidified atmosphere of 95% air and 5% CO2. Cells were fixed with 4% paraformaldehyde (Sigma, MO, USA) for 20 min at room temperature after the incubation with simvastatin (1 mg/ml; Merk, Japan) for 24 h or NGF (50 ng/ml, Biomedical Technologies, MA, USA) for 72 h. We also prepared the same conditions with NOS inhibitor, Nnitro-L-arginine methyl ester (L-NAME; Sigma, MO, USA). Then the fixed cells were incubated overnight at 4°C with anti-peripherin monoclonal antibody (Chemicon International, CA, USA), and then they were stained according to the biotin-streptavidin horseradish peroxidase method (Sensi TekTM HRP, Scytek, UT, USA). Cell division was determined by measuring the cell number by a Coulter Counter ZBI (Coulter Electronics, Health, FL, USA). For this measurement, each cell culture was
Fig. 2. NO production in PC12 cells treated with simvastatin (24 h) or NGF (72 h). (a) Cells were treated with simvastatin in a dose-dependent fashion (1, 5 and 10 mg/ml). The cells treated with simvastatin (10 mg/ml) were concomitantly treated with NOS inhibitors (L-NAME, 2 mM; L-NMMA, 2 mM). (b) Cells were treated with NGF (50 ng/ml) with or without 2 mM of L-NAME. The values of NO production in the control cells represents as 100%. The amount of NOx in the control medium was 3.53 ± 0.78 pmol. Data are the mean ± SEM of three to four cultures. *P , 0.05, compared to the control; **P , 0.05, compared to the medium treated with NGF (50 ng/ml); *P , 0.05, compared to the medium treated with simvastatin (10 mg/ml).
Fig. 1. Immunostaining with anti-peripherin antibody in simvastatintreated (1 mg/ml, 24 h) PC12 cells with or without 2 mM of L-NAME and in NGF-treated (50 ng/ml, 72 h) PC12 cells with or without 2 mM of L-NAME. The expression of peripherin is evident in neurites in both cases (b,c,e). (a) Control, (b) NGF, (c) Simvastatin, (d) NGF and LNAME, (e) Simvastatin and L-NAME. Scale bar, 100 mm.
washed with a phosphate-buffered saline. A mixture of 0.5 ml of 0.05% trypsin and 0.05% EDTA was then used to get a cell suspension. The reactions were terminated by adding 0.5 ml of the growth medium to the cell suspension. NO released from PC12 cells was measured as its NOx metabolites (nitrate, nitrite) in the medium, by means of a chemiluminescence detector (Sievers Instruments, CO, USA). A standard curve was constructed using 50–250 pmol NaNO3 for calculation of picomolar NOx content per sample. The background signal in the medium was subtracted from each measured value. NO production was calculated as follows: NO production = test − blank/cell number (105 cells), the blank was the NOx metabolites
M. Sano et al. / Neuroscience Letters 243 (1998) 73–76
(nitrate, nitrite) from DMEM supplemented with serum. NO production in the control culture was expressed as 100%. Data are expressed as the mean ± SEM, and were analyzed by one-way analysis of variance followed by Fisher’s test to correct for multiple comparisons of treatment. Fig. 1 shows the light microscopic observation of the PC12 cells immunostained with anti-peripherin antibody. Simvastatin-treatment caused the cells to become round with long neurites. The neurites outgrown from the cells treated with both simvastatin and NGF were confirmed to contain peripherin protein (Fig. 1b,c). When these cells were concomitantly treated with 2 mM of L-NAME, the cells treated with NGF did not show neurite formation while the cells with simvastatin did (Fig. 1d,e). Fig. 2a shows NO production in the simvastatin-treated PC12 cells for 24 h. Cells were plated at the density of 3 × 105 cells/ml. The cells were treated with simvastatin in a dose-dependent fashion (1, 5, 10 mg/ml). The cells treated with simvastatin at a concentration of 10 mg/ml were also treated with one of NOS inhibitors concomitantly. L-NAME (2 mM) and of N-monomethyl-L-arginine (L-NMMA; 2 mM; Sigma, MO, USA) were used as NOS inhibitors. Simvastatin treatment increased NO production in PC12 cells in a dose-dependent fashion, and NOS inhibitors significantly suppressed the increase. Fig. 2b shows the data on the NGF treatment (50 ng/ml) for 72 h. In this case, cells were plated at the density of 105 cells/ml for the longer period (72 h) of the culture. L-NAME suppressed the increase in NO production in the PC12 cells treated with NGF. The control medium contained 3.53 ± 0.78 pmol of NOx. Fig. 3 shows the effects of simvastatin treatment on the division of PC12 cells. Cells were treated with simvastatin (10 mg/ml) with or without NOS inhibitors (L-NAME, 2 mM; L-NMMA, 2 mM) for 24 h. The growth was remark-
Fig. 3. The effects of simvastatin on the division of PC12 cells. The number of cells was counted 24 h after the treatment with simvastatin (10 mg/ml) with or without NOS inhibitors (L-NAME, 2 mM; LNMMA, 2 mM). Data are the mean ± SEM for three to four cultures. *P , 0.05, compared to the control.
75
ably inhibited by the simvastatin treatment and NOS inhibitors could not rescue it. In the parallel experiment with NGF (50 ng/ml, 72 h) instead of simvastatin, the decrease in the cell number was limited. However, in the case of NGF, L-NAME (2 mM) recovered the decrease in the cell number as previously reported [11] (data not shown). In our previous study, we found that simvastatin induced neurite-like outgrowth in PC12 cells [14], but we did not examine whether the outgrowth was accompanied by peripherin expression. In the present study, the expression of peripherin was clearly demonstrated in the neurite-like outgrowth of PC12 cells treated with simvastatin. Thus we confirmed that simvastatin caused real neurite outgrowth in PC12 cells. Peripherin is a protein which is expressed when neuronal cells differentiate under such condition as NGF treatment [3]. Our data shows that the neurite outgrowth of PC12 cells treated with simvastatin is the same as that shown in NGF treated PC12 cells from the viewpoint of peripherin formation. However, there are several differences in the neurite outgrowth in PC12 cells between simvastatin and general neurotrophic factors such as NGF. Firstly, simvastatin can induce the neurite outgrowth rapidly, i.e. within only 20 h from the start of simvastatin treatment [14], while in the case of NGF, it takes at least 2 days to get neurite-outgrowth in PC12 cells [3]. Secondly, the shape of simvastatin-treated cells is different from that of NGF-treated ones. Simvastatin causes formation of a roundish cell body while NGF treated cells remain polygonal. Simvastatin treated patients are sometimes known to be suffering from central nervous system (CNS) adverse effects [2,4,13,15]. The neuronal cells may be damaged in some way by the simvastatin treatment. In our present study simvastatin inhibited the cell division of PC12 cells. The inhibition of neuronal cell division could be related to the CNS adverse effects. A recent study demonstrates that NO is involved in the cytostatic effect of NGF on PC12 cells [11]. They claimed that both NO and NGF were necessary to be present at the same time to induce differentiation and arrest cell cycle in PC12 cells because in iNOS (−/−) mutant cells NO donor alone or NGF alone failed to induce differentiation and arrest cell cycle in the cells [11]. In this study we showed that NO from NGF-treated PC12 cells was involved in the differentiation of PC12 cells because NOS inhibitor inhibited NGF induced growth arrest and cell differentiation. We first thought that NO from simvastatin-treated PC12 cells was also involved in the differentiation of PC12 cells. However, NO had nothing to do with differentiation in simvastatin-treated PC12 cells, because NOS inhibitors could not abolish simvastatin induced PC12 cell differentiation. So we speculate that the mechanism by which simvastatin induces cytostatic action and differentiation is different from those induced by NGF. Although the mechanisms by which simvastatin stimulates NO production in PC12 cells are still unclear, simvas-
76
M. Sano et al. / Neuroscience Letters 243 (1998) 73–76
tatin may induce NOS. In our preliminary experiments, simvastatin-treated PC12 cells were stained with anti-NOS antibody (data not shown). Furthermore, it has been reported that membrane cholesterol contents affect uptake capacity of calcium ion in cells [1,5]. Although we did not examine whether PC12 cells treated with simvastatin are enhanced in the Ca2+ uptake activity, the Ca2+ may activate Ca2+-dependent NOS activities such as eNOS or nNOS activity. Lovastatin induces cyclin-dependent kinase inhibitors (P21, P27) in human breast cancer cells in a cell cycleindependent fashion [7,8]. Cell cycle phase transitions in eukaryotic cells are driven by the regulation of the activity of protein kinases known as cyclin-dependent kinases (Cdk). p27 is redistributed from cyclin D1/Cdk4 to cyclin A/Cdk2 complexes in Swiss 3T3 mouse fibroblast cells treated with lovastatin [12]. It is likely that a similar principle of redistribution of p27 is seen in other instances of cell cycle arrest. Therefore HMG Co-A reductase may be the key enzyme which correlates to the mechanisms of cell division and differentiation. Although the reason why NO is not involved in the all static action and differentiation is unclear in simvastatin-treated cells, HMG-CoA reductase inhibitor may be a single differentiation factor that is not effected by NO. In conclusion, we demonstrate that simvastatin induces NO production in PC12 cells in a dose-dependent fashion. The neurite outgrown in simvastatin-treated PC12 cells is similar to the one seen in NGF-treated PC12 cells, but the mechanism of NO production, growth arrest and differentiation induced by these two substances are different. Further study is required to make these mechanisms clear. The authors thank Dr. Toshiyuki Himi, Tokyo Medical and Dental University, for helpful suggestions and critical discussion of the manuscript. We also thank Dr. Hitoshi Wakisaka, National Defense Medical College, for kindly providing PC12 cells. [1] Bialecki, RA. and Tulenko, TN., Excess membrane cholesterol alters calcium channels in arterial smooth muscle, Am. J. Physiol., 257 (1989) C306–C314.
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