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Cloning and functional studies of a splice variant of CYP26B1 expressed in vascular cells
Ali Ateia Elmabsout1, Ashok Kumawat, Patricia Saenz-Méndez
1Department of Clinical Medicine, School of Health Sciences, Örebro University, Örebro, Sweden.
Background:
All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene.
Methodology/Principal Findings:
The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells.
Conclusions/Significance:
Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.
Insights
A newly identified CYP26B1 gene variant, lacking exon 2, is found in vascular cells and elevated in atherosclerosis. This variant degrades all-trans retinoic acid (atRA) slower than the full-length enzyme, impacting cardiovascular health.
Area of Science:
- Molecular Biology
- Cardiovascular Science
- Gene Expression Regulation
Background:
- All-trans retinoic acid (atRA) is crucial for cardiovascular development but implicated in diseases like atherosclerosis.
- Cellular atRA levels are tightly regulated by cytochrome P450 enzymes (CYPs), with CYP26 potentially key in vascular cells.
- This study focuses on a spliced variant of CYP26B1, a regulator of atRA metabolism.
Purpose of the Study:
- To molecularly clone, characterize, and determine the function of an atRA-induced spliced variant of the CYP26B1 gene.
- To investigate the expression and role of this spliced variant in vascular cells and atherosclerotic lesions.
Main Methods:
- Amplification and sequencing of the spliced CYP26B1 variant from atRA-treated human aortic smooth muscle cells.
- Analysis of CYP26B1 variant expression in cultured vascular cells and human arterial tissues (normal and atherosclerotic).
- Functional assays involving transfection of cells with the spliced variant to assess atRA catabolism.
Main Results:
- A spliced CYP26B1 variant lacking exon 2 was identified and found expressed in human endothelial and smooth muscle cells, as well as in normal and atherosclerotic arteries.
- Both full-length and spliced CYP26B1 variants were induced by atRA in cultured vascular cells.
- Spliced CYP26B1 mRNA transcripts were significantly higher (4.5-fold) in atherosclerotic lesions compared to normal arteries and showed a 20-fold increase upon atRA treatment.
- The spliced variant degrades atRA at one-third the rate of the full-length enzyme, with transfection studies showing variable effects on atRA catabolism.
Conclusions:
- Vascular cells express a spliced CYP26B1 variant (lacking exon 2), which is upregulated in atherosclerotic lesions.
- This spliced variant exhibits reduced all-trans retinoic acid degradation capacity compared to the full-length enzyme.
- Further research is required to elucidate the precise substrate specificity and functional significance of the CYP26B1 splice variant in vascular health and disease.
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