Abstract Title

The expression of the HSP family in dental pulp stem cells within adult teeth treated with nicotine

Lead Author Affiliation

Doctor of Dental Surgery

Lead Author Status

DDS Year 2

Expected Graduation Date

2020

Presentation Category

Research

Introduction/Context/Diagnosis

The heat shock protein (HSP) family includes many factors produced during the human response to infection, toxins, hypoxia, inflammation, and other stressors. HSP act as chaperones to new proteins by stabilizing structures during protein folding and by preventing undesirable protein aggregation. While HSP plays a role in the protecting the human body from stress, they can also contribute to the survival of cancerous cells possibly by attempting to block apoptosis and other cellular mechanisms. Thus, HSP inhibitors could act as potential therapeutic agents for certain cancers. As stem cell therapies are developed, HSPs could play an important role in helping stem cells both survive and maintain their differentiability during dental trauma. As a result, HSP therapy may promote pulpal regeneration and serve as an alternative to endodontic therapy. Hsp90, Hsp70, and Hsp27 are the primary heat shock proteins located in the cytoplasm of eukaryotic cells. While these all have functions in cytoprotection and the cell survival pathway, Hsp90, the largest of these three, has also been shown to play a role in intracellular signaling and cell-cycle control. Additionally, pHsp27 and Hsp27 have also been linked to cellular and potentially tumor growth within dental tissues. Changes in Hsp27 and Hsp70 expression also occur during apoptosis in pulp tissues after cavity preparation, so these proteins may play an inhibitory role in the apoptotic pathway. Together, these heat shock proteins are thought to function in the survival of dental pulps under stress. Through this experiment, we show that dental pulp stem cells (DPSC) initially express high levels of HSPs when expose to nicotine, a toxin, but HSP expression is later reduced when exposed to high concentrations of nicotine.

Methods/Treatment Plan

Cell Culture

Human DPSCs were purchased from Lonza. Cells before passage 10 were used for experiments. DPSCs were cultured as previously reported8. Briefly, DPSCs were cultured in DMEM (Gibco) supplemented with 10% FBS, antibiotics and ascorbic acids.

Western blot

DPSCs were cultured in 60-mm dish and incubated with nicotine (Sigma) at different concentrations (100nM-10mM) for up to 24 hours. Cells were then collected at 1 hour, 6 hours and 24 hours with RIPA buffer. 50ug total protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked and probed with antibodies:HSP90, HSP70, HSP27, p-HSP27-S78, and tubulin (Abcam). Images were captured with Gel Doc System (Biorad) and quantification was done with image J.

Image Quantification

Quantification was done with ImageJ using the gel analysis routine. Nine equal-sized rectangular sections were analyzed, and the relative density of contents was plotted. The straight line function was used to enclose the plotted peaks, and the wand tool was used to quantify the bands. The quantified information was analyzed in Excel, normalized to β-tubulin and then normalized to the control during each experiment.

Statistics

Data were expressed as SD. Student’s t-tests (2-tailed) were used to compare the data. P ≤ 0.05 was considered to be significant.

Results/Outcome

Hsp27 treated with 10 uM nicotine exhibited significantly increased expression after one hour of treatment. Hsp90 and Hsp70 also exhibited higher than average values after the first hour for most concentrations but to statistically insignificant values. This one-hour time point is relevant because the peak plasma concentration of nicotine in smokers occurs after one hour at a level of 22.5 ± 17.01ng/ml. Nicotine dosing in this experiment begins at around 17 ng/ml. In comparison, at the six-hour time point only Hsp90 was significantly different when treated with 1 uM and 1 mM nicotine. However, while Hsp90 levels increased at 1 hour when treated with 1 uM nicotine in comparison to the control, these levels decreased significantly after 6 hours of the same treatment. This illustrates how cellular response changes with response to time due to the alternative functionality of each protein. Additionally, Hsp90, Hsp70, and Hsp27 exhibited reduced expression after a twenty-four-hour treatment of 10 mM nicotine, while pHsp27 exhibited significantly higher expression at the same concentration and time point. At this same time point and high concentration of nicotine, β-tubulin levels were greatly reduced. This indicates that the cells began to die due to extreme stress.

Significance/Conclusions

Through this experiment, we found that some HSP bands increased concentration shortly after exposure to the noxious stimulus; however, the relative levels of all HSP except for pHSP27 dropped over larger segments of time significantly. Future experiments should include analysis of the expression of other apoptotic factors and visualization of protein aggregates following DPSC trauma. Localization of HSP and induction of apoptosis might also be studied during and after cellular trauma. Another future experiment could investigate if added HSP could contribute to protein re-folding and pulpal regeneration through luminescence studies. Additionally, Hsp90 has been implicated in the stability tyrosine hydroxylase during periods of chronic alcohol and drug abuse. It would be interesting to test whether Hsp90 and other heat shock proteins play a similar in nicotine abusers by studying the levels of expression in DPSCs from long term smokers and non-smokers.

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The expression of the HSP family in dental pulp stem cells within adult teeth treated with nicotine

The heat shock protein (HSP) family includes many factors produced during the human response to infection, toxins, hypoxia, inflammation, and other stressors. HSP act as chaperones to new proteins by stabilizing structures during protein folding and by preventing undesirable protein aggregation. While HSP plays a role in the protecting the human body from stress, they can also contribute to the survival of cancerous cells possibly by attempting to block apoptosis and other cellular mechanisms. Thus, HSP inhibitors could act as potential therapeutic agents for certain cancers. As stem cell therapies are developed, HSPs could play an important role in helping stem cells both survive and maintain their differentiability during dental trauma. As a result, HSP therapy may promote pulpal regeneration and serve as an alternative to endodontic therapy. Hsp90, Hsp70, and Hsp27 are the primary heat shock proteins located in the cytoplasm of eukaryotic cells. While these all have functions in cytoprotection and the cell survival pathway, Hsp90, the largest of these three, has also been shown to play a role in intracellular signaling and cell-cycle control. Additionally, pHsp27 and Hsp27 have also been linked to cellular and potentially tumor growth within dental tissues. Changes in Hsp27 and Hsp70 expression also occur during apoptosis in pulp tissues after cavity preparation, so these proteins may play an inhibitory role in the apoptotic pathway. Together, these heat shock proteins are thought to function in the survival of dental pulps under stress. Through this experiment, we show that dental pulp stem cells (DPSC) initially express high levels of HSPs when expose to nicotine, a toxin, but HSP expression is later reduced when exposed to high concentrations of nicotine.