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KEAP1-NRF2-IKKβ crosstalk protects periodontal ligament fibroblasts subjected to mechanical vibratory forces
Swathi Jayan1, Jeena John1, Madhav Manoj K1
1Department of Orthodontics, Sri Sankara Dental College, Akathumuri, Varkala, Trivandrum, Kerala State, India.
Objectives:
When cells experience acute trauma from forces, cytoprotective molecules such as NRF2 and activation of inflammatory pathways led by NFkB are synthesized and released. An action-binding protein, KEAP1, plays a central role in these activities. The present study aimed to determine the effect of differential magnitudes of vibratory mechanical force application on the KEAP1 molecule and its downstream regulators, such as NRF2 and IKKβ.
Design:
A fibroblast cell culture was performed on periodontal ligament cells scraped from atraumatically extracted premolar teeth. These cells were subjected to low (50 Hz), medium, (100 Hz) high (150 Hz), and very high (200 Hz) vibratory settings for 20 min at 0, 24, and 48 h. mRNA and protein analysis of KEAP1, NRF2, and IKKβ was performed using real-time PCR, indirect ELISA test, and Western blot, respectively.
Results:
The results indicated that vibratory mechanical force application interfered with cultured periodontal ligament fibroblasts' normal growth and homeostasis. While KEAP1's mRNA expression and protein activity were downregulated, NRF2's and IKKβ's were upregulated. As force magnitude increases, KEAP1's downregulation increases, while NRF2 and IKKβ's upregulation continues.
Conclusions:
The study revealed an inverse relationship between KEAP1, NRF2, and IKKβ in periodontal ligament fibroblasts subjected to mechanical forces. A decrease in KEAP1 and an increase in NRF2 and IKKβ mRNA and protein levels to different vibrational frequencies can evoke cytoprotective responses in the cells.
Insights
Mechanical forces on periodontal ligament cells decrease KEAP1 (Kelch-like ECH-associated protein 1) and increase NRF2 (Nuclear factor erythroid 2-related factor 2) and IKKβ (Inhibitor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta). This suggests a cytoprotective response to vibratory stimuli.
Area of Science:
- Cellular Biology
- Biomechanical Engineering
- Molecular Biology
Background:
- Cellular trauma triggers cytoprotective molecules like NRF2 and inflammatory pathways via NFkB.
- KEAP1 (Kelch-like ECH-associated protein 1) is a key regulator in these cellular responses.
- Understanding KEAP1's role is crucial for cellular protection mechanisms.
Purpose of the Study:
- To investigate the impact of varying vibratory mechanical force magnitudes on KEAP1.
- To analyze the downstream effects on NRF2 and IKKβ.
- To determine the relationship between mechanical force and these key regulatory molecules.
Main Methods:
- Periodontal ligament fibroblasts were cultured.
- Cells were exposed to distinct vibratory frequencies (50-200 Hz) for 20 minutes at set intervals.
- KEAP1, NRF2, and IKKβ expression and activity were quantified using RT-PCR, ELISA, and Western blot.
Main Results:
- Vibratory mechanical forces disrupted normal fibroblast growth and homeostasis.
- KEAP1 mRNA and protein levels were significantly downregulated.
- NRF2 and IKKβ mRNA and protein levels were upregulated, with increased force magnitude amplifying these effects.
Conclusions:
- An inverse relationship exists between KEAP1 and NRF2/IKKβ in periodontal ligament fibroblasts under mechanical stress.
- Decreased KEAP1 and increased NRF2/IKKβ indicate a cellular cytoprotective response to mechanical forces.
- These findings highlight the role of KEAP1, NRF2, and IKKβ in cellular adaptation to mechanical stimuli.
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