Nicotinic acetylcholine receptor activation completely blocks osteoclastogenesis by interfering with osteoclast precursor differentiation in vitro (original) (raw)
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Nicotinic acetylcholine receptors modulate osteoclastogenesis
Arthritis Research & Therapy, 2016
Background: Our aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis. Methods: The presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity. Results: We could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca 2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine. Conclusions: We have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo.
Nicotinic modulation of gene expression in osteoblast cells, MG-63
Bone, 2011
Exposure to nicotine causes a broad range of biological and molecular effects on osteoblasts which are known to play a crucial role in bone metabolism and fracture healing. Most effects of nicotine on the osteoblasts are long-term adaptations at the genomic level. To identify the nicotine-regulated genes, the Agilent technologies whole human genome gene expression microarray was performed on RNA samples from osteoblast-like cells, MG-63, exposed to 100 μM nicotine. Repeat and cross-controlled microarray analyses revealed 842 genes whose expression was consistently altered at P b 0.05 level following nicotine treatment. Gene ontology analysis suggested effects of nicotine on various biological and cellular processes which were associated with survival, proliferation, differentiation and apoptosis processes within the cell. Quantitative real-time reverse transcriptase PCR analysis confirmed altered expression in 7 out of 9 genes tested. The identified genes tested in the current study support our previous report that nicotine regulates the expression of genes that promote osteoblast proliferation and/or anti-apoptosis processes. Furthermore, using nicotinic acetylcholine receptor antagonists blocked the majority of the nicotine effects, indicating that these changes are dependent on nAChR activation. These results established a novel and consistent nicotinic activation of nAChR in osteoblast cells which has a broad role affecting cellular physiology through modulation of gene expression.
Nicotine Stimulates Osteoclast Resorption in a Porcine Marrow Cell Model
Journal of Periodontology, 2003
Background: Combustible tobacco use is generally linked with accelerated periodontal bone loss and diminished post-surgical wound healing; however, the pathogenesis of this process at the cellular level remains unclear. Nicotine is known to affect human gingival fibroblast orientation, attachment, and β 1 integrin expression, yet little is known about its effects on osteoclasts, the cells most responsible for bone resorption. The purpose of this study was to determine the effects of physiologically relevant nicotine levels on porcine osteoclast function as measured by resorption of calcium phosphate. Methods: Pure nicotine was diluted in medium to the following concentrations: 0.03 µM, 0.15 µM, 0.30 µM, 0.60 µM, and 1.50 µM. Porcine osteoclasts were seeded onto calcium phosphate multi-test slides and incubated at 37°C with half media changes every 24 hours. Cells received 0, 0.15, 0.30, 0.60, and 1.50 µM nicotine, or 25 nM parathyroid hormone (PTH). Osteoclast resorption was quantified by measuring the resorbed surface area of the calcium phosphate substrate. Results: Osteoclast cultures resorbed bone slices and calcium phosphate substrate. All nicotine concentrations and PTH resulted in statistically significantly greater mean percent resorptions than the control group (P <0.05). However, no statistical difference was found between the various nicotine doses or PTH. The number of osteoclasts increased in a linear relationship to the increasing nicotine concentrations; however, no correlation was found between osteoclast number and the amount of resorption. Conclusions: Nicotine is non-toxic to osteoclasts at the clinically relevant levels tested. Nicotine appears to stimulate osteoclast differentiation and resorption of calcium phosphate, the major component of bone. Nicotine-modulated osteoclast stimulation may, in part, explain the increased rapidity of periodontal bone loss and refractory disease incidence in smokers.
Global spine journal, 2012
Previous studies by our group showed that nicotine delivered via a transdermal nicotine patch significantly enhanced posterior spinal fusion rates in rabbits. Nicotine transdermal patches provide a steady serum level; there may be a dose-dependent effect of nicotine on posterior spinal fusion. In an in vitro cell culture model of rabbit bone marrow-derived osteoblast-like cells, cells were exposed to different concentrations of nicotine (0, 20, 40, 80 ng/mL and 10, 100, 250 μg/mL). Wells were stained with an alkaline phosphatase (ALP) staining kit to determine ALP enzyme activity. Cells were stained with Von Kossa for mineralization. A two-way analysis of variance (ANOVA) using dose and time as variables showed significant differences among groups; post hoc analysis showed that the 100-μg/mL dose of nicotine significantly enhanced ALP activity over controls. A one-way ANOVA using dose as the variable showed that the 100- and 250-μg/mL doses had significantly greater mineralization t...
Nicotine induced proliferation and cytokine release in osteoblastic cells
International Journal of Molecular Medicine, 2006
Smoking has deleterious effects on osteoporosis and periodontitis both characterized by bone loss. Smoking also interferes with the protective effect that hormone replacement therapy (HRT) has on bone loss. Our study investigated two mechanisms by which smoking may affect bone metabolism: nicotine-induced proliferation and nicotine-induced cytokine secretion in osteoblasts. Two osteoblastic cell models were used: mouse osteoblasts derived from mouse calvaria and human osteoblasts. Thymidine incorporation and immunoassays were used to evaluate proliferation, interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) secretion. Parametric and nonparametric statistical analyses were used for comparisons. The results showed that nicotine induced stimulation and inhibition of proliferation in both osteoblastic cell models. In human osteoblasts, the proliferative and inhibitory effects were also donor dependent. Il-6 secretion showed different patterns in mouse and human osteoblasts. In mouse osteoblasts, nicotine significantly increased IL-6 secretion and estradiol significantly inhibited the nicotine-induced IL-6 release. In human osteoblasts, cells derived from one subject did not respond to nicotine. However, in the second sample, nicotine increased secretion of Il-6 but estradiol did not oppose this effect. In human osteoblasts, nicotine also induced an increase in the TNF-alpha secretion and estradiol opposed this increase. These results suggest that nicotine affects bone metabolism by modulating proliferation, and Il-6 and TNF-alpha secretion. These studies provide a possible explanation for differences in bone loss among subjects who smoke and offer a possible mechanism for the oppositional effect of smoking on HRT in subjects with bone loss.
Effects of nicotine on markers of bone turnover in ovariectomized rats
Pan African Medical Journal
Introduction: osteoporosis is characterized by low bone mass and density, as well as change in microarchitecture of bone tissue leading to decreased bone strength. In vitro research shows nicotine can increase osteoblast activity and proliferation, also suppress osteoclast activity. Therefore we explore nicotine anti-resorptive property by in vivo true experimental and randomized posttest only controlled group research that was conducted in 18-20 weeks old Rattus norvegicus. Methods: twenty-five female rats were divided into five groups, with 5 rats per group. The first group represented normal rats (Sham), while the second to fifth group underwent bilateral ovariectomy. The second group serves as positive control group (ovariectomy-only/OVX). The third to fifth group serve as dose 1 (P1-0.25mg/kg), dose 2 (P2-0.5 mg/kg), and Dose 3 (P3-0.75 mg/kg) treatment group receiving daily per-oral nicotine for 28 days, started 3 weeks post-ovariectomy. After 28 days treatment, the serum was checked. Results: nicotine has dose-dependent manner on serum osteocalcin and serum DPD level. Level of osteocalcin in P2 group was significantly lower (Mann-Whitney, p = 0.008) compared to OVX group (59.4% lower). Level of DPD in all group was not significantly different (ANOVA, p < 0.05) but shows lowest level in P2 group. For serum calcitonin level, there's no significant different between groups. Conclusion: nicotine at right low-dose might be able to inhibit osteoclast activity, thus open a possibility of anti-resorptive property of nicotine.
Effect of nicotine on bone healing in rats - A histological study
2014
Background & Objectives: Nicotine is the major alkaloid in tobacco products ( Nicotiona tabacum ) and a psychoactive ingredient responsible for the Central Nervous System (CNS) effects and tobacco addiction. It's been reported to have effects directly on the small blood vessels in producing vasoconstriction and increased vascular resistance that exerts on the microvasculature inhibiting the angioblastic response during re- vascularization and limits the recruitment of cytokines, Bone Morphogenic Proteins (BMPs), Transforming Growth Factor - � (TGF - �), Platelet Derived Growth Factor (PDGF) and the basic Fibroblast Growth Factor (FGF). This leads to inhibition of re-epithelialization, osteogenesis and cellular healing. This study intends to demonstrate histologically the effect of nicotine on bone healing and the healing of bone defects incorporated with autogenous bone graft in an animal model. Methods: 60 female Wistar rats were used in the study. Nicotine hemisulfate at a dos...
Differential Effects of Nicotine and Smoke Condensate on Bone Cell Metabolic Activity
Journal of Orthopaedic Trauma, 2005
Objective: Delayed or impaired healing of skeletal trauma in patients who smoke has been attributed to vascular responses of nicotine absorption and/or a direct effect of nicotine or other smoke components on bone cells. In vivo studies indicate variability in osteosynthetic response to nicotine versus smoke inhalation. We tested the hypothesis that components of cigarette smoke other than nicotine may be responsible for the adverse skeletal effects of smoking. Design: In vitro cultures of MC3T3-E1 osteoblastlike cells were exposed to varying doses of nicotine or condensates of cigarette smoke. Metabolic assays included alkaline phosphatase activity, collagen synthesis, and total protein synthesis as well as cell proliferation. Results: Variations in the degree of response were noted between bone cell preparations. Nicotine elicited a significant dose-dependent stimulation of bone cell metabolism in all studies. This was detected as increases in alkaline phosphatase activity and increases in total protein and collagen synthesis. Responses were noted with nicotine doses as low as 12.5 ng/mL (half the nicotine level circulating in smokers). In one study, maximum stimulation occurred at 250 ng/mL with levels reaching 74% (total protein) and 104% (collagen) greater than control cultures. In a second study, 222% and 627% stimulation of protein and collagen synthesis over controls was noted using 100 ng/mL. Addition of the nicotine receptor antagonist mecamylamine reduced the nicotine stimulation. Preparations of smoke condensate with equivalent nicotine concentrations reduced all indices of metabolic activity. Cell proliferation was stimulated by both nicotine (20-25%) and smoke condensate (38-46%). Conclusion: The data suggest that nicotine acts as a direct stimulant of bone cell metabolic activity. Smoke condensate containing equivalent levels of nicotine elicits an inhibitory effect. A probable speculation is that the delay in clinical healing of skeletal trauma in smoking patients may in part be a result of absorption of components of smoke other than nicotine.