Nicotine modulates bone metabolism-associated gene expression in osteoblast cells
Abstract:
Smoking can affect risks of osteoporosis, fracture healing time, and nonunion rates. This paper attempts to assess the effects of smoking through the action of nicotine on cell proliferation and gene expression. Low levels of nicotine upregulated cell proliferation and osteocalcin, type I collagen, and alkaline phosphatase gene expression. High nicotine levels associated with that of heavy smokers had the opposite effect. D-tubocurarine inhibited these effects and leads to the conclusion that nicotine acts via the nicotinic acetylcholine receptor (nAChR).
Introduction:
The many systemic effects smoking has on the body are of special interest to orthopaedic surgeons dealing with bone fracture. Nicotine is the main component of over 150 known toxic compounds. This study hypothesizes that nicotine acts through binding to the nAChR, causing upregulation or downregulation of osteoblast regulatory genes suppressing osteogenesis, promoting bone resorption, and delaying osteoblast differentiation.
Materials and Methods:
The MG-63 cell line was used as the osteoblasts in this study. A cell proliferation assay was done with varying concentrations of nicotine and cell numbers determined in 24 hour increments up for 72 hours. RT-PCR was run for osteocalcin, type I collagen, and alkaline phosphatase. All experiments were performed in triplicate at least.
Results:
See publication.
Discussion:
The levels of nicotine tested were chosen because they correlate to levels in the blood of habitual smokers and saliva levels of long-term snuff users. There was a bimodal effect on cell proliferation: low levels increased cell proliferation, whereas high levels dramatically decreased proliferation ending in cell death. The three genes chosen are involved in bone metabolism. The nicotine-induced increase in cell proliferation was inhibited by high levels of D-tubocurarine, a nCHhR antagonist.
This paper aims to determine the effect of nicotine on human osteoblast-like cells. Specifically, it studies the changes in cell proliferation and gene expression of human osteosarcoma cells (MG63). The genes that were looked at were Type I Collagen, alkaline phosphatase, and osteocalcin, which all play a part in the mediation of bone metabolism. In addition, the mechanism through which these changes occur is looked into. The paper has a nice, informative introduction that discusses why this study is of interest: smoking has many systemic effects that are important to orthopaedic surgeons. Previous studies have shown that smoking delays the rate of fracture healing, and induce an increased risk of osteoporosis and non-union.
The authors do a good job of using relevant concentrations of nicotine in their experiments. They varied concentrations of nicotine (0, 0.01, 0.1, 1, 10, 100, 1,000, and 10,000 micro molar) according to levels found in the blood and saliva of smokers and snuff users, respectively. The incubation was also varied from 1 to 72 hours, which was good, although I would have liked to see a longer term incubation to compare. They also use a nicotinic acetylcholine receptor antagonist (dTC) in order to see if the nicotine acted at the cellular level by binding to nicotinic acetylcholine receptor of osteoblast cells. They vary the concentration of dTC as well.
In general, all numbers included a mean with a standard deviation. All graphs were well-presented and included clear, relevant legends/labels. Since they used nicotine concentrations that correlated to those of habitual smokers, the results can be easily interpreted. My main complaint is that they don't really explain the interesting result for the lower concentrations of nicotine: namely that cell proliferation increases and gene expression was up-regulated. The conclusions say simply that nicotine decreases bone metabolism in habitual smokers, but it isn't really clear if both up-regulation and down-regulation would decrease bone metabolism. If not, then the conclusions don't explain the results for low nicotine levels well enough. Something else that is not elaborated upon enough is the fact that dTC alone exhibited a slightly toxic effect on cell proliferation. Other than that, the paper was well put-together and didn't give too much unnecessary information.
The last paragraph gives a nice summary of what can be taken away from this study and how it can potentially be of use in the future. The authors speculate that their results, along with future studies, may lead to new treatments to speed the healing process in smokers and patients with bone injuries in general.
6 comments:
Great job summarizing the paper. I am surprised there is a correlation between smoking and fracture healing times, but am especially surprised by the opposite effect when comparing low levels of nicotine and higher levels. It is easy to understand the negative effects of having poor bone healing at higher levels of nicotine, but what are the implications for lighter smokers who according to the study, would have higher bone healing rates – are there negative results to that as well?
As noted in the previous comments, it's rather odd how low nicotine levels promote growth! So the take home message is if you fracture a bone, start smoking but only one cigarette a day :) Although this would be ridiculous, using nicotine patches at the site of injury could be used to speed bone growth while minimizing the other negative effects of nicotine by localizing its release. The potential use of nicotine in this scenario should be explored further.
Future research could also directly study nicotine's effect on bone cells. Although this research showed that nicotine acts directly on the nicotinic acetylcholine receptor, there must be additional factors that can explain its positive effect at low concentrations and negative effect at high concentrations. The complex interactions of the cells with nicotine should be investigated to shed light into this interesting phenomena.
I am curious about a number of things regarding this paper. Firstly, EGF is a bioactive molecule that is meant to induce protein kinase phosphorylation so I am wondering if whether their results were based on or normalized for current existing levels within the normal body in order to better visualize differences cause by varying the concentration of EGF from those found standard in living systems. I am also cautious about the western blotting results as they seem to have a high level of noise and so I do not see a strong justification for their conclusion, I feel the assay should have been rerun in order to verify results with a cleaner blot.
Hey Hinesh. I think your comment might be for another paper since this one did not deal with EGF or have a western blot.
Interesting that nicotine can affect bone healing rates! However, I am not convinced by the authors' argument here. While the authors cell proliferation assays in Figure 1 showed a decrease in cell viability at higher concentrations, one can reach this effect using any toxic compound. This effect may not be limited to nicotine. Furthermore, RT-PCR results in Figure 3 showed the same pattern of decreased RNA expression at high nicotine concentrations. However, could such a decrease reflect overall RNA levels rather than RNA levels in bone-specific genes? I did not see any convincing controls. I believe that the upregulation of RNA expression and cell viability at low nicotine levels is the more interesting finding here. I definitely agree with Eric that the authors' should look more into this result.
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