- Email: [email protected]
Nicotine in cigarettes promotes chromogranin A production by human periodontal ligament fibroblasts
archives of oral biology 58 (2013) 1029–1033
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Nicotine in cigarettes promotes chromogranin A production by human periodontal ligament fibroblasts Sunao Sadaoka a,*, Kimitoshi Yagami b, Shigeru Maki a a b
Department of Oral Health, School of Dentistry, Matsumoto Dental University, Shiojiri, Nagano, Japan Department of Social Dentistry, School of Dentistry, Matsumoto Dental University, Shiojiri, Nagano, Japan
article info
abstract
Article history:
Objective: The body produces chromogranin A (ChgA) in response to stress as an adaptive
Accepted 26 February 2013
reaction. While ChgA is used as an index of autonomic nervous system activity, it is also
Keywords:
with periodontal disease and cigarette smokers has been reported. However, its production
Nicotine
in periodontal tissue cells subjected to stress and its immunomodulatory action have not
involved in the immunomodulation system, and an increase in its production in patients
Chromogranin A
been clarified. To investigate the influence of nicotine on periodontal tissue, we measured
Human periodontal ligament
ChgA production in nicotine-treated periodontal ligament fibroblasts.
fibroblast (HPdLF)
Design: Using normal human periodontal ligament-derived fibroblasts (HPdLF) as a peri-
Active and passive smoking
odontal tissue model, untreated cells (control) and cells treated with 10 and 100 nM nicotine sulfate corresponding to passive and active cigarette smoking, respectively, were cultured for a specific time. The ChgA level in the culture fluid was measured as ChgA production in HPdLF employing ELISA. ChgA gene expression was quantified employing qPCR. In addition, intracellular localisation was confirmed by immunohistochemical staining. Results: In the control HPdLF group, a low level of ChgA was produced, and immunohistochemical ChgA-positive reactions were observed in the nucleus and cytoplasm. In the nicotine-treated HPdLF group, the ChgA mRNA expression level, protein production, and immunostaining-positive rate increased, and the levels were higher in the cells treated with 10 nM nicotine corresponding to passive smoking than in the cells treated with 100 nM nicotine corresponding to active smoking. Conclusion: Human periodontal ligament-derived fibroblasts (HPdLF) produced ChgA, and nicotine increased ChgA production. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The inflammatory response of periodontitis is caused by stress that affects the immune system.1 There are two types of stress: micro-stress in organelles and macro-stress at the systemic level or mentally.2,3 We looked at whether tobacco can be a micro/macro-stress factor for the progression of periodontitis. Nicotine intake by active and passive smoking is
involved in the development and progression of periodontal disease, but its mechanism has not yet been elucidated.3–7 Nicotine contained in tobacco acts on the periodontal tissue cells and has been reported to promote the production of inflammatory cytokines: IL-1b and LPS-induced TNF-a, as well as to induce changes in the cell cycle and differentiation marker values.6–9 Nicotine as micro-stress inhibits the growth of human periodontal ligament-derived fibroblasts (HPdLF).10 Passive smoking induces mental stress and influences the
* Corresponding author at: 1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan. Tel.: +81 263 51 2153; fax: +81 263 2223. E-mail addresses: [email protected], [email protected] (S. Sadaoka). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.02.012
1030
archives of oral biology 58 (2013) 1029–1033
immune pathway of the autonomic nerve system, in addition to inhalation of toxic substances contained in tobacco smoke.11 The mechanisms behind the links between mental stress and periodontitis are believed to be alterations of endocrine and immunological responses.10 The neuroendocrine system cells produce chromogranin A (ChgA), which is a physiologically active substance that acts in response to mental stress.12 ChgA is a physiologically active substance of the neuroendocrine system and serves as an index of autonomic nerve system activity.12 It is also related to the systemic and local immunomodulation systems, being involved in antimicrobial peptide production, mast cell migration, leukotriene and prostaglandin production, and histamine release.13–15 Epidemiologically, ChgA has been reported to be associated with periodontal and smokingrelated airway diseases, and ChgA is abundant in saliva and blood of patients.16,17 ChgA is a stress marker that covers from micro- to macro-stress in vivo. No cell-level analysis of ChgA production in fibroblasts or the influence of nicotine in tobacco on ChgA has been reported. In this study, to clarify the reactions of normal human periodontal ligament-derived fibroblasts (HPdLF) to nicotine, we investigated ChgA production in response to nicotine at concentrations corresponding to active and passive smoking.
2.
Materials and methods
2.1.
Cell culture
Normal human periodontal ligament-derived fibroblasts (HPdLF, Lonza, Basel, Switzerland) were cultured as periodontal tissue cells in stromal cell basal medium (SCBM, Lonza, Basel, Switzerland) supplemented with Single Quots (CC-4181; hFGF-B, insulin, 0.5% FBS, 50 mg/ml gentamycin, 50 mg/ml amphotericin B, Lonza, Basel, Switzerland) following the attached instructions. Cells were passaged in the logarithmic growth phase up to 3 generations. For nicotine added to culture medium, 5% nicotine sulfate (C10H14N21/2 H2SO4, Torii Pharmaceutical Co., Ltd., Tokyo, Japan) was used.
2.2.
Preparation of smoking cell model
HPdLF was cultured in Single Quots-containing SCBM at a cell density of 1 105 cells/ml, and confluent cells were used in the experiment (n = 6). Regarding cells untreated with nicotine sulfate (untreated group) as a control, 10 and 100 nM nicotine sulfate corresponding to passive and active smoking, respectively, was added to the culture medium, and the cells were cultured for 12 h,18 following the procedure reported by Nishida et al.5
2.3.
Measurement of ChgA production
Total protein was collected from culture fluid using the RC/DC protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). The ChgA level in protein was measured by ELISA using the Human Chromogranin A EIA kit (Yanaihara Institute Inc., Tokyo, Japan) (n = 6).
2.4.
DNA measurement
DNA was collected from cultured cells using TRIzol1 (Life Technologies Corp., Carlsbad, CA, USA) and measured using Nanodrop 2000 (Thermo Fisher Science Inc., Wilmington, DE, USA).
2.5.
Semi-quantitative PCR
After processing using the TaqMan1 Gene Expression Cells-toCTTM Kit, cells were combined with TaqMan1 Gene Expression Assays (Life Technologies Corp., CA, USA), ChgA probes (Assay ID: Hs00900373_m1), and glyceraldehyde 3-phosphate dehydrogenase probes (GAPDH, Assay ID: Hs03929097_g1) and subjected to PCR. mRNA expression was investigated by relative quantitative analysis employing the DDCt method, performed using the Applied Step One plus PCR System (n = 6).
2.6.
Immunohistochemical staining
For indirect immunostaining, the Histofine SAB-PO(R) Kit and AEC substrate kit as a colour development substrate (Nichirei Bio Systems, Tokyo, Japan) were used. Cells were fixed in 4% paraformaldehyde solution (pH 7.2) at room temperature for 5 min. After washing with PBS, the cells were reacted with anti-human ChgA polyclonal antibodies (Nichirei Bio Systems, Tokyo, Japan) as the primary antibody at room temperature for one hour. The negative control was reacted with rabbit normal serum.
2.7.
Statistical analysis
Data were analysed using Student’s t-test, and a p-value lower than 0.05 was regarded as significant (*p < 0.05, **p < 0.01).
3.
Results
3.1. Changes in the ChgA production level in the smoking model (Fig. 1) Compared with that in the control, ChgA secretion significantly increased in the HPdLF smoking model ( p < 0.05). The ChgA production level was significantly higher in the 10 nM than in the 100 nM nicotine sulfate-treated group.
3.2. Changes in the ChgA gene expression level in the smoking model (Fig. 2) Compared with that in the control, about 1.2- and 2.0-fold increases in the ChgA gene expression level were noted in the HPdLF smoking model when cells were treated with 100 and 10 nM nicotine sulfate, respectively, and significant differences were noted between all combinations of groups.
3.3. Intracellular localisation of ChgA in the smoking model (Fig. 3) Weakly positive reactions with anti-ChgA antibodies were noted in the nucleus and cytoplasm even in the control HPdLF.
archives of oral biology 58 (2013) 1029–1033
Fig. 1 – Measurement of the ChgA level in culture supernatant. Compared with that in the control group, ChgA secretion increased in the nicotine-treated group. The increase was greater in the group treated with 10 nM nicotine, corresponding to passive smoking, than in the group treated with 100 nM nicotine, corresponding to active smoking. Significant differences were noted between all combinations of groups.
1031
Fig. 2 – ChgA mRNA expression. Compared with that in the control group, about 1.2- and 2.0-fold increases in the ChgA gene expression level were noted when cells were treated with 100 and 10 nM nicotine sulfate, corresponding to active and passive smoking, respectively. Significant differences were noted between all combinations of groups.
Fig. 3 – Immunohistochemical staining of ChgA and the number of positive cells. Nicotine increased cells showing positive reactions with anti-ChgA antibodies in the nucleus and cytoplasm (A). Significant differences in the positive cell rate from that in the control were noted in the nicotine-treated groups. However, the difference between the 10 nM and 100 nM nicotine-treated groups was not significant (B).
1032
archives of oral biology 58 (2013) 1029–1033
Nicotine sulfate increased the number of cells showing positive reactions in the nucleus and cytoplasm, and a significant difference was noted in the positive cell rate between the control and each nicotine-treated group. However, the 10 nM nicotine-treated group between 100 nM nicotinetreated group was not significant ( p = 0.07).
Ethical approval Not required.
Acknowledgement 4.
Discussion
4.1. ChgA production in HPdLF and nicotine-induced changes in the production level It was clarified that HPdLF constitutively produces ChgA, and nicotine increases the production level. However, no cell-level analysis of nicotine-induced changes in ChgA production or involvement of fibroblast-derived ChgA in the immune system has been reported. Van der Pauw et al.19 reported that HPdLF constituting most of the periodontal ligament has a mechanical stress-sensing system. Assuming that HPdLF also has a system sensing nicotine as microstress, ChgA production in HPdLF may increase as a response. It was recognised that nicotine as a stress factor and ChgA were extremely high in association with periodontitis.
4.2.
Significance of ChgA expression in HPdLF
ChgA was expressed in fibroblasts, which has not previously been reported, and was present in the nucleus and cytoplasm. ChgA serves as a modulatory factor of oxidative stress and apoptosis by controlling scavenger receptor-mediated NOS production in non-neural cells.20 A ChgA fragment, catestatin, has cell-proliferative and growth factor actions, an apoptosisinhibitory action,20,21 and inflammation-regulating action including vasodilation.15,22 Another ChgA fragment, vasostatin, has been reported to serve as a factor controlling the adhesion of fibroblasts to adjacent cells,23 suggesting that ChgA produced by periodontal ligament fibroblasts also has similar actions.
5.
Conclusion
It was clarified that periodontal ligament fibroblasts produce ChgA, and nicotine increases ChgA production. The ChgA production level was higher in cells treated with 10 nM nicotine, corresponding to passive smoking, than in those treated with 100 nM nicotine, corresponding to active smoking.
Funding None.
Competing interests None declared.
This study was supported by JSPS Grant-in-Aid for Scientific Research 23593112.
references
1. Rettori E, De Laurentiis A, Zorrilla Zubilete M, Rettori V, Elverdin JC. Anti-inflammatory effect of the endocannabinoid anandamide in experimental periodontitis and stress in the rat. Neuroimmunomodulation 2012;19:293–303. 2. Li CL, Lu HK. Mental stress and its association with periodontal health. Journal of Dental Sciences 2006;1:155–60. 3. Lee SI, Kang KL, Shin SI, Herr Y, Lee YM, Kim EC. Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells. Journal of Periodontal Research 2012;47:299–308. 4. Walter C, Kaye EK, Dietrich T. Active and passive smoking: assessment issues in periodontal research. Periodontol 2012;58:84–92. 2000. 5. Nishida N, Yamamoto Y, Tanaka M, Maeda K, Kataoka K, Nakayama K, et al. Association between passive smoking and salivary markers related to periodontitis. Journal of Clinical Periodontology 2006;33:717–23. 6. Wang XJ, Liu YF, Wang QY, Tsuruoka M, Ohta K, Wu SX, et al. Functional expression of alpha 7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues. Cell and Tissue Research 2010;340:347–55. 7. Breivik T, Gundersen Y, Gjermo P, von Ho¨rsten S. Opstad PK.Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis. Journal of Periodontal Research 2009;44:110–6. 8. Liu YF, Ge X, Wen LY, Wang XJ. Targeting nicotinic acetylcholine receptor to treat smoking-related periodontitis. Medical Hypotheses 2011;76:244–5. 9. Arredondo J, Nguyen VT, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. A receptor-mediated mechanism of nicotine toxicity in oral keratinocytes. Laboratory Investigation 2001;81:1653–68. 10. James JA, Sayers NM, Drucker DB, Hull PS. Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts. Journal of Periodontology 1999;70:518–25. 11. Hamer M, Stamatakis E, Batty GD. Objectively assessed secondhand smoke exposure and mental health in adults: cross-sectional and prospective evidence from the Scottish Health Survey. Archives of General Psychiatry 2010;67: 850–5. 12. Nakane H. Salivary chromogranin A as index of psychosomatic stress response. Technical Journal R&D Review 1999;34:17–22. 13. Hoq MI, Niyonsaba F, Ushio H, Aung G, Okumura K, Ogawa H. Human catestatin enhances migration and proliferation of normal human epidermal keratinocytes. Journal of Dermatological Science 2011;64:108–18. 14. Radek KA, Elias PM, Taupenot L, Mahata SK, O’Connor DT, Gallo RL. Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by
archives of oral biology 58 (2013) 1029–1033
15.
16.
17.
18.
19.
suppressing antimicrobial peptide production. Cell Host Microbe 2010;7:277–89. Aung G, Niyonsaba F, Ushio H, Kajiwara N, Saito H, Ikeda S, et al. Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines. Immunology 2011;132:527–39. Hironaka M, Ansai T, Soh I, Ishisaka A, Awano S, Yoshida A, et al. Association of salivary levels of cortisol and dehydroepiandrosterone with periodontitis in older japanese adults. Biomedical Research 2008;29:125–30. Sørhaug S, Langhammer A, Waldum HL, Hveem K, Steinshamn S. Increased serum levels of chromogranin A in male smokers with airway obstruction. European Respiratory Journal 2006;28:542–8. Takeuchi H, Kubota S, Murakashi E, Zhou Y, Endo K, Ng PS, et al. Nicotine-induced CCN2: from smoking to periodontal fibrosis. Journal of Dental Research 2010;89:34–9. van der Pauw MT, Klein-Nulend J, van den Bos T, Burger EH, Everts V, Beertsen W. Response of periodontal ligament fibroblasts and gingival fibroblasts to pulsating fluid flow: nitric oxide and prostaglandin E2 release and expression of
20.
21.
22.
23.
1033
tissue non-specific alkaline phosphatase activity. Journal of Periodontal Research 2000;35:335–43. Hooper C, Fry VA, Sevastou IG, Pocock JM. Scavenger receptor control of chromogranin A-induced microglial stress and neurotoxic cascades. FEBS Letters 2009;583: 3461–6. Theurl M, Schgoer W, Albrecht K, Jeschke J, Egger M, Beer AG, et al. The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factordependent mechanism. Circulation Research 2010;107: 1326–35. Angelone T, Quintieri AM, Brar BK, Limchaiyawat PT, Tota B, Mahata SK, et al. The antihypertensive chromogranin a peptide catestatin acts as a novel endocrine/paracrine modulator of cardiac inotropism and lusitropism. Endocrinology 2008;149: 4780–93. Gasparri A, Sidoli A, Sanchez LP, Longhi R, Siccardi AG, Marchisio PC, et al. Chromogranin A fragments modulate cell adhesion. Identification and characterisation of a proadhesive domain. Journal of Biological Chemistry 1997;272:20835–43.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Nicotine in cigarettes promotes chromogranin A production by human periodontal ligament fibroblasts Sunao Sadaoka a,*, Kimitoshi Yagami b, Shigeru Maki a a b
Department of Oral Health, School of Dentistry, Matsumoto Dental University, Shiojiri, Nagano, Japan Department of Social Dentistry, School of Dentistry, Matsumoto Dental University, Shiojiri, Nagano, Japan
article info
abstract
Article history:
Objective: The body produces chromogranin A (ChgA) in response to stress as an adaptive
Accepted 26 February 2013
reaction. While ChgA is used as an index of autonomic nervous system activity, it is also
Keywords:
with periodontal disease and cigarette smokers has been reported. However, its production
Nicotine
in periodontal tissue cells subjected to stress and its immunomodulatory action have not
involved in the immunomodulation system, and an increase in its production in patients
Chromogranin A
been clarified. To investigate the influence of nicotine on periodontal tissue, we measured
Human periodontal ligament
ChgA production in nicotine-treated periodontal ligament fibroblasts.
fibroblast (HPdLF)
Design: Using normal human periodontal ligament-derived fibroblasts (HPdLF) as a peri-
Active and passive smoking
odontal tissue model, untreated cells (control) and cells treated with 10 and 100 nM nicotine sulfate corresponding to passive and active cigarette smoking, respectively, were cultured for a specific time. The ChgA level in the culture fluid was measured as ChgA production in HPdLF employing ELISA. ChgA gene expression was quantified employing qPCR. In addition, intracellular localisation was confirmed by immunohistochemical staining. Results: In the control HPdLF group, a low level of ChgA was produced, and immunohistochemical ChgA-positive reactions were observed in the nucleus and cytoplasm. In the nicotine-treated HPdLF group, the ChgA mRNA expression level, protein production, and immunostaining-positive rate increased, and the levels were higher in the cells treated with 10 nM nicotine corresponding to passive smoking than in the cells treated with 100 nM nicotine corresponding to active smoking. Conclusion: Human periodontal ligament-derived fibroblasts (HPdLF) produced ChgA, and nicotine increased ChgA production. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The inflammatory response of periodontitis is caused by stress that affects the immune system.1 There are two types of stress: micro-stress in organelles and macro-stress at the systemic level or mentally.2,3 We looked at whether tobacco can be a micro/macro-stress factor for the progression of periodontitis. Nicotine intake by active and passive smoking is
involved in the development and progression of periodontal disease, but its mechanism has not yet been elucidated.3–7 Nicotine contained in tobacco acts on the periodontal tissue cells and has been reported to promote the production of inflammatory cytokines: IL-1b and LPS-induced TNF-a, as well as to induce changes in the cell cycle and differentiation marker values.6–9 Nicotine as micro-stress inhibits the growth of human periodontal ligament-derived fibroblasts (HPdLF).10 Passive smoking induces mental stress and influences the
* Corresponding author at: 1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan. Tel.: +81 263 51 2153; fax: +81 263 2223. E-mail addresses: [email protected], [email protected] (S. Sadaoka). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.02.012
1030
archives of oral biology 58 (2013) 1029–1033
immune pathway of the autonomic nerve system, in addition to inhalation of toxic substances contained in tobacco smoke.11 The mechanisms behind the links between mental stress and periodontitis are believed to be alterations of endocrine and immunological responses.10 The neuroendocrine system cells produce chromogranin A (ChgA), which is a physiologically active substance that acts in response to mental stress.12 ChgA is a physiologically active substance of the neuroendocrine system and serves as an index of autonomic nerve system activity.12 It is also related to the systemic and local immunomodulation systems, being involved in antimicrobial peptide production, mast cell migration, leukotriene and prostaglandin production, and histamine release.13–15 Epidemiologically, ChgA has been reported to be associated with periodontal and smokingrelated airway diseases, and ChgA is abundant in saliva and blood of patients.16,17 ChgA is a stress marker that covers from micro- to macro-stress in vivo. No cell-level analysis of ChgA production in fibroblasts or the influence of nicotine in tobacco on ChgA has been reported. In this study, to clarify the reactions of normal human periodontal ligament-derived fibroblasts (HPdLF) to nicotine, we investigated ChgA production in response to nicotine at concentrations corresponding to active and passive smoking.
2.
Materials and methods
2.1.
Cell culture
Normal human periodontal ligament-derived fibroblasts (HPdLF, Lonza, Basel, Switzerland) were cultured as periodontal tissue cells in stromal cell basal medium (SCBM, Lonza, Basel, Switzerland) supplemented with Single Quots (CC-4181; hFGF-B, insulin, 0.5% FBS, 50 mg/ml gentamycin, 50 mg/ml amphotericin B, Lonza, Basel, Switzerland) following the attached instructions. Cells were passaged in the logarithmic growth phase up to 3 generations. For nicotine added to culture medium, 5% nicotine sulfate (C10H14N21/2 H2SO4, Torii Pharmaceutical Co., Ltd., Tokyo, Japan) was used.
2.2.
Preparation of smoking cell model
HPdLF was cultured in Single Quots-containing SCBM at a cell density of 1 105 cells/ml, and confluent cells were used in the experiment (n = 6). Regarding cells untreated with nicotine sulfate (untreated group) as a control, 10 and 100 nM nicotine sulfate corresponding to passive and active smoking, respectively, was added to the culture medium, and the cells were cultured for 12 h,18 following the procedure reported by Nishida et al.5
2.3.
Measurement of ChgA production
Total protein was collected from culture fluid using the RC/DC protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). The ChgA level in protein was measured by ELISA using the Human Chromogranin A EIA kit (Yanaihara Institute Inc., Tokyo, Japan) (n = 6).
2.4.
DNA measurement
DNA was collected from cultured cells using TRIzol1 (Life Technologies Corp., Carlsbad, CA, USA) and measured using Nanodrop 2000 (Thermo Fisher Science Inc., Wilmington, DE, USA).
2.5.
Semi-quantitative PCR
After processing using the TaqMan1 Gene Expression Cells-toCTTM Kit, cells were combined with TaqMan1 Gene Expression Assays (Life Technologies Corp., CA, USA), ChgA probes (Assay ID: Hs00900373_m1), and glyceraldehyde 3-phosphate dehydrogenase probes (GAPDH, Assay ID: Hs03929097_g1) and subjected to PCR. mRNA expression was investigated by relative quantitative analysis employing the DDCt method, performed using the Applied Step One plus PCR System (n = 6).
2.6.
Immunohistochemical staining
For indirect immunostaining, the Histofine SAB-PO(R) Kit and AEC substrate kit as a colour development substrate (Nichirei Bio Systems, Tokyo, Japan) were used. Cells were fixed in 4% paraformaldehyde solution (pH 7.2) at room temperature for 5 min. After washing with PBS, the cells were reacted with anti-human ChgA polyclonal antibodies (Nichirei Bio Systems, Tokyo, Japan) as the primary antibody at room temperature for one hour. The negative control was reacted with rabbit normal serum.
2.7.
Statistical analysis
Data were analysed using Student’s t-test, and a p-value lower than 0.05 was regarded as significant (*p < 0.05, **p < 0.01).
3.
Results
3.1. Changes in the ChgA production level in the smoking model (Fig. 1) Compared with that in the control, ChgA secretion significantly increased in the HPdLF smoking model ( p < 0.05). The ChgA production level was significantly higher in the 10 nM than in the 100 nM nicotine sulfate-treated group.
3.2. Changes in the ChgA gene expression level in the smoking model (Fig. 2) Compared with that in the control, about 1.2- and 2.0-fold increases in the ChgA gene expression level were noted in the HPdLF smoking model when cells were treated with 100 and 10 nM nicotine sulfate, respectively, and significant differences were noted between all combinations of groups.
3.3. Intracellular localisation of ChgA in the smoking model (Fig. 3) Weakly positive reactions with anti-ChgA antibodies were noted in the nucleus and cytoplasm even in the control HPdLF.
archives of oral biology 58 (2013) 1029–1033
Fig. 1 – Measurement of the ChgA level in culture supernatant. Compared with that in the control group, ChgA secretion increased in the nicotine-treated group. The increase was greater in the group treated with 10 nM nicotine, corresponding to passive smoking, than in the group treated with 100 nM nicotine, corresponding to active smoking. Significant differences were noted between all combinations of groups.
1031
Fig. 2 – ChgA mRNA expression. Compared with that in the control group, about 1.2- and 2.0-fold increases in the ChgA gene expression level were noted when cells were treated with 100 and 10 nM nicotine sulfate, corresponding to active and passive smoking, respectively. Significant differences were noted between all combinations of groups.
Fig. 3 – Immunohistochemical staining of ChgA and the number of positive cells. Nicotine increased cells showing positive reactions with anti-ChgA antibodies in the nucleus and cytoplasm (A). Significant differences in the positive cell rate from that in the control were noted in the nicotine-treated groups. However, the difference between the 10 nM and 100 nM nicotine-treated groups was not significant (B).
1032
archives of oral biology 58 (2013) 1029–1033
Nicotine sulfate increased the number of cells showing positive reactions in the nucleus and cytoplasm, and a significant difference was noted in the positive cell rate between the control and each nicotine-treated group. However, the 10 nM nicotine-treated group between 100 nM nicotinetreated group was not significant ( p = 0.07).
Ethical approval Not required.
Acknowledgement 4.
Discussion
4.1. ChgA production in HPdLF and nicotine-induced changes in the production level It was clarified that HPdLF constitutively produces ChgA, and nicotine increases the production level. However, no cell-level analysis of nicotine-induced changes in ChgA production or involvement of fibroblast-derived ChgA in the immune system has been reported. Van der Pauw et al.19 reported that HPdLF constituting most of the periodontal ligament has a mechanical stress-sensing system. Assuming that HPdLF also has a system sensing nicotine as microstress, ChgA production in HPdLF may increase as a response. It was recognised that nicotine as a stress factor and ChgA were extremely high in association with periodontitis.
4.2.
Significance of ChgA expression in HPdLF
ChgA was expressed in fibroblasts, which has not previously been reported, and was present in the nucleus and cytoplasm. ChgA serves as a modulatory factor of oxidative stress and apoptosis by controlling scavenger receptor-mediated NOS production in non-neural cells.20 A ChgA fragment, catestatin, has cell-proliferative and growth factor actions, an apoptosisinhibitory action,20,21 and inflammation-regulating action including vasodilation.15,22 Another ChgA fragment, vasostatin, has been reported to serve as a factor controlling the adhesion of fibroblasts to adjacent cells,23 suggesting that ChgA produced by periodontal ligament fibroblasts also has similar actions.
5.
Conclusion
It was clarified that periodontal ligament fibroblasts produce ChgA, and nicotine increases ChgA production. The ChgA production level was higher in cells treated with 10 nM nicotine, corresponding to passive smoking, than in those treated with 100 nM nicotine, corresponding to active smoking.
Funding None.
Competing interests None declared.
This study was supported by JSPS Grant-in-Aid for Scientific Research 23593112.
references
1. Rettori E, De Laurentiis A, Zorrilla Zubilete M, Rettori V, Elverdin JC. Anti-inflammatory effect of the endocannabinoid anandamide in experimental periodontitis and stress in the rat. Neuroimmunomodulation 2012;19:293–303. 2. Li CL, Lu HK. Mental stress and its association with periodontal health. Journal of Dental Sciences 2006;1:155–60. 3. Lee SI, Kang KL, Shin SI, Herr Y, Lee YM, Kim EC. Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells. Journal of Periodontal Research 2012;47:299–308. 4. Walter C, Kaye EK, Dietrich T. Active and passive smoking: assessment issues in periodontal research. Periodontol 2012;58:84–92. 2000. 5. Nishida N, Yamamoto Y, Tanaka M, Maeda K, Kataoka K, Nakayama K, et al. Association between passive smoking and salivary markers related to periodontitis. Journal of Clinical Periodontology 2006;33:717–23. 6. Wang XJ, Liu YF, Wang QY, Tsuruoka M, Ohta K, Wu SX, et al. Functional expression of alpha 7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues. Cell and Tissue Research 2010;340:347–55. 7. Breivik T, Gundersen Y, Gjermo P, von Ho¨rsten S. Opstad PK.Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis. Journal of Periodontal Research 2009;44:110–6. 8. Liu YF, Ge X, Wen LY, Wang XJ. Targeting nicotinic acetylcholine receptor to treat smoking-related periodontitis. Medical Hypotheses 2011;76:244–5. 9. Arredondo J, Nguyen VT, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. A receptor-mediated mechanism of nicotine toxicity in oral keratinocytes. Laboratory Investigation 2001;81:1653–68. 10. James JA, Sayers NM, Drucker DB, Hull PS. Effects of tobacco products on the attachment and growth of periodontal ligament fibroblasts. Journal of Periodontology 1999;70:518–25. 11. Hamer M, Stamatakis E, Batty GD. Objectively assessed secondhand smoke exposure and mental health in adults: cross-sectional and prospective evidence from the Scottish Health Survey. Archives of General Psychiatry 2010;67: 850–5. 12. Nakane H. Salivary chromogranin A as index of psychosomatic stress response. Technical Journal R&D Review 1999;34:17–22. 13. Hoq MI, Niyonsaba F, Ushio H, Aung G, Okumura K, Ogawa H. Human catestatin enhances migration and proliferation of normal human epidermal keratinocytes. Journal of Dermatological Science 2011;64:108–18. 14. Radek KA, Elias PM, Taupenot L, Mahata SK, O’Connor DT, Gallo RL. Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by
archives of oral biology 58 (2013) 1029–1033
15.
16.
17.
18.
19.
suppressing antimicrobial peptide production. Cell Host Microbe 2010;7:277–89. Aung G, Niyonsaba F, Ushio H, Kajiwara N, Saito H, Ikeda S, et al. Catestatin, a neuroendocrine antimicrobial peptide, induces human mast cell migration, degranulation and production of cytokines and chemokines. Immunology 2011;132:527–39. Hironaka M, Ansai T, Soh I, Ishisaka A, Awano S, Yoshida A, et al. Association of salivary levels of cortisol and dehydroepiandrosterone with periodontitis in older japanese adults. Biomedical Research 2008;29:125–30. Sørhaug S, Langhammer A, Waldum HL, Hveem K, Steinshamn S. Increased serum levels of chromogranin A in male smokers with airway obstruction. European Respiratory Journal 2006;28:542–8. Takeuchi H, Kubota S, Murakashi E, Zhou Y, Endo K, Ng PS, et al. Nicotine-induced CCN2: from smoking to periodontal fibrosis. Journal of Dental Research 2010;89:34–9. van der Pauw MT, Klein-Nulend J, van den Bos T, Burger EH, Everts V, Beertsen W. Response of periodontal ligament fibroblasts and gingival fibroblasts to pulsating fluid flow: nitric oxide and prostaglandin E2 release and expression of
20.
21.
22.
23.
1033
tissue non-specific alkaline phosphatase activity. Journal of Periodontal Research 2000;35:335–43. Hooper C, Fry VA, Sevastou IG, Pocock JM. Scavenger receptor control of chromogranin A-induced microglial stress and neurotoxic cascades. FEBS Letters 2009;583: 3461–6. Theurl M, Schgoer W, Albrecht K, Jeschke J, Egger M, Beer AG, et al. The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factordependent mechanism. Circulation Research 2010;107: 1326–35. Angelone T, Quintieri AM, Brar BK, Limchaiyawat PT, Tota B, Mahata SK, et al. The antihypertensive chromogranin a peptide catestatin acts as a novel endocrine/paracrine modulator of cardiac inotropism and lusitropism. Endocrinology 2008;149: 4780–93. Gasparri A, Sidoli A, Sanchez LP, Longhi R, Siccardi AG, Marchisio PC, et al. Chromogranin A fragments modulate cell adhesion. Identification and characterisation of a proadhesive domain. Journal of Biological Chemistry 1997;272:20835–43.