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[Effect of mm-LDL on NF-kB activation in endothelial cell]
1Department of Pathology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Objective:
To investigate the signal transduction pathway of NF-kB activated by minimally modified low density lipoprotein (mm-LDL) in endothelial cells and the effect of NF-kB on platelet derived growth factor b (PDGFb) mRNA expression.
Methods:
mm-LDL was prepared through iron oxidation by dialyzing the native LDL against FeSO4 in PBS. Endothelial cells were incubated in a medium containing mm-LDL, TNF, and IL-1 respectively and electrophoretic mobility shift assay (EMSA) was displayed to check on the activation of NF-kB. Luciferase reporter gene was analysed to investigate the effect of nuclear factor inducing kinase (NIK), inhibitor of NF-kB kinase alpha (IKK alpha) and inhibitor of NF-kB kinase beta (IKK beta) on NF-kB activation. In addition, endothelial cells were transfected using PDGFb promoter-luciferase for reporter gene analysis or transfected with mut-NIK for slot blot analysis to study the effect of NF-kB on PDGFb mRNA expression.
Results:
mm-LDL was able to activate NF-kB in endothelial cells. mut-NIK and mut-IKK beta inhibited luciferase activity induced by mm-LDL. mm-LDL could also enhance luciferase activity controlled by upstream sequence of PDGFb promoter which contains element interacting with NF-kB. Result of slot blot showed inhibition of PDGFb mRNA expression by mut-NIK in the endothelial cells stimulated by mm-LDL.
Conclusion:
mm-LDL may activate NF-kB through NIK-IKK beta pathway and promote PDGFb mRNA expression in endothelial cells.
Insights
Minimally modified low-density lipoprotein (mm-LDL) activates nuclear factor kappa B (NF-kB) in endothelial cells. This activation, mediated by the NIK-IKK beta pathway, promotes platelet-derived growth factor b (PDGFb) mRNA expression.
Area of Science:
- Molecular Biology
- Cell Biology
- Cardiovascular Research
Background:
- Endothelial cells play a crucial role in vascular health.
- Minimally modified low-density lipoprotein (mm-LDL) is implicated in atherosclerosis.
- Nuclear factor kappa B (NF-kB) is a key transcription factor involved in inflammatory responses.
Purpose of the Study:
- To elucidate the NF-kB signal transduction pathway activated by mm-LDL in endothelial cells.
- To investigate the impact of NF-kB activation on platelet-derived growth factor b (PDGFb) mRNA expression.
Main Methods:
- Preparation of mm-LDL via iron oxidation.
- Electrophoretic mobility shift assay (EMSA) to assess NF-kB activation.
- Luciferase reporter gene assays to study NIK, IKK alpha, and IKK beta involvement.
- Reporter gene analysis and slot blot analysis to evaluate PDGFb mRNA expression.
Main Results:
- mm-LDL effectively activated NF-kB in endothelial cells.
- Mutant NIK (mut-NIK) and mutant IKK beta (mut-IKK beta) inhibited mm-LDL-induced luciferase activity.
- mm-LDL enhanced luciferase activity controlled by the PDGFb promoter, indicating NF-kB interaction.
- mut-NIK inhibited PDGFb mRNA expression in mm-LDL-stimulated endothelial cells.
Conclusions:
- mm-LDL activates NF-kB through the NIK-IKK beta pathway in endothelial cells.
- This activation leads to the promotion of PDGFb mRNA expression.
- The findings provide insights into the molecular mechanisms underlying mm-LDL-induced vascular changes.