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Expression and alternative splicing of fibronectin mRNA in human diploid endothelial cells during aging in vitro
F Pagani1, L Zagato, J A Maier
1Fondazione Rivetti, Laboratory of Biochemistry and Molecular Biology, Milan, Italy.
Abstract:
Different mRNAs for fibronectin arise from the variable processing of a single primary transcript. We used ribonuclease protection assay to investigate the changes occurring in fibronectin expression and the alternative splicing of mRNA precursor during aging in vitro of human diploid endothelial cells. Senescent endothelial cells release more protein and contain 4-5-fold more fibronectin mRNA than young cells. The pattern of alternative splicing of fibronectin mRNA, with the EDA and the CS1 segments largely included (35% and 77%, respectively) and the EDB segment undetectable, correlates well with previous studies at the protein level both in vitro and in vivo. No changes in the splicing pattern of fibronectin mRNA precursor were detected during endothelial cellular senescence. The increased expression of fibronectin in senescent cells may be a result of the activity of interleukin-1 alpha, which is overexpressed in senescent endothelial cells. It could be also important in vivo during aging and in atherosclerotic lesions.
Insights
Senescent endothelial cells exhibit increased fibronectin mRNA and protein levels. Alternative splicing patterns of fibronectin mRNA remain unchanged during cellular aging, suggesting post-transcriptional regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Aging Research
Background:
- Fibronectin (FN) is a crucial extracellular matrix protein involved in cell adhesion, migration, and tissue repair.
- Differential mRNA processing leads to various fibronectin isoforms, impacting its function.
- Cellular senescence, a state of irreversible growth arrest, is associated with altered gene expression and extracellular matrix remodeling.
Purpose of the Study:
- To investigate changes in fibronectin mRNA expression and alternative splicing during in vitro aging of human diploid endothelial cells.
- To determine if fibronectin mRNA splicing patterns are altered in senescent endothelial cells.
- To explore potential mechanisms underlying increased fibronectin expression in aging cells.
Main Methods:
- Ribonuclease protection assay (RPA) was employed to quantify fibronectin mRNA levels.
- Analysis of alternative splicing patterns of fibronectin mRNA precursor was performed.
- Comparison of fibronectin expression and splicing in young versus senescent endothelial cells.
Main Results:
- Senescent endothelial cells displayed a 4-5 fold increase in fibronectin mRNA levels compared to young cells.
- The alternative splicing pattern of fibronectin mRNA, characterized by inclusion of EDA and CS1 segments and exclusion of EDB, was consistent in both young and senescent cells.
- No significant changes in fibronectin mRNA precursor splicing were observed during endothelial cellular senescence.
Conclusions:
- Increased fibronectin expression in senescent endothelial cells is not due to altered alternative splicing of its mRNA.
- The elevated fibronectin levels in aging cells may be regulated at the transcriptional level or influenced by factors like interleukin-1 alpha.
- These findings have implications for understanding fibronectin's role in aging and the development of atherosclerotic lesions.