Cloning and Functional Studies of a Splice Variant of CYP26B1 Expressed in Vascular Cells

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

The role of the pyridoxine (vitamin B6) biosynthesis enzyme PDX1 in ultraviolet-B radiation responses in plants

Ultraviolet-B radiation regulates plant growth and morphology at low and ambient fluence rates but can severely impact on plants at higher doses. Some plant UV-B responses are related to the formation of reactive oxygen species (ROS) and pyridoxine (vitamin B6) has been reported to be a quencher of ROS. UV-B irradiation of Arabidopsis Col-0 plants resulted in increased levels of PDX1 protein, compared with UV-A-exposed plants. This was shown by immunoblot analysis using specific polyclonal antibodies raised against the recombinant PDX1.3 protein and confirmed by mass spectrometry analysis of immunoprecipitated PDX1. The protein was located mainly in the cytosol but also to a small extent in the membrane fraction of plant leaves. Immunohistochemical analysis performed in pea revealed that PDX1 is present in UV-B-exposed leaf mesophyll and palisade parenchyma but not in epidermal cells. Pyridoxine production increased in Col-0 plants exposed to 3 days of UV-B, whereas in an Arabidopsis pdx1.3 mutant UV-B did not induce pyridoxine biosynthesis. In gene expression studies performed after UV-B exposure, the pdx1.3 mutant showed elevated transcript levels for the LHCB1*3 gene (encoding a chlorophyll a/b-binding protein of the photosystem II light-harvesting antenna complex) and the pathogenesis-related protein 5 (PR-5) gene, compared with wild type.This is a pre-print article</p

mRNA Levels for the CYP26B1 Spliced Variant in Vascular Cells and in Normal vessels.

<p><i>CYP26B1</i> expression in human umbilical vein endothelial cells (HUVECs), aortic smooth muscle cells (AOSMCs) and normal kidney arteries. PCR was performed using primers annealing to exon 1 and exon 3 of <i>CYP26B1</i>. In the presence of exon 2 the expected PCR products is 438 bp, in its absence only 213 bp. (A) HUVEC, (B) AOSMC, (C) three independent samples of normal kidney arteries, (D) quantitative RT-PCR analysis of <i>CYP26B1</i> transcript levels in atherosclerotic lesion (<i>n = 6</i>) and normal kidney arteries (<i>n = 5</i>) and transcript levels of CYP26B1 splice variant in atherosclerotic lesion treated with DMSO (n = 4) and atRA (n = 4) (right panel).</p

Amino acid sequence of the splice variant <i>CYP26B1</i>.

<p>Alignment of full length and spliced <i>CYP26B1</i> reveals the exclusion of sequence corresponding to exon2 in the latter protein.</p

Protein expression of the CYP26B1 splice variant in HUVEC, AOSMCs and COS-1 cells.

<p>(A1) Expression of full length and spliced CYP26B1 in atRA-treated human umbilical vein endothelial cells (HUVECs) and (A2) aortic smooth muscle cells (AOSMCs) treated with atRA (lane 1) and DMSO (lane 2) and COS-1 cells transfected with constructs for spliced variant (A1, lane 3, and A2, lane 4) and full length CYP26B1 (A1, lane 4, and A2, lane 3), detected with Western blot analysis. (B) Cell-based reporter pRARE-TA-SEAP was used to measure retinoid levels in the presence of the full length and spliced CYP26B1 in COS-1 cells. SEAP: luciferase ratio in COS-1 cells transfected with either of the two different <i>CYP26B1</i> variants, both variants simultaneously, or empty vector after 24 hours treatment with either atRA or DMSO (control). (C1) Cellular concentration of exogenously added [³H] atRA in COS-1 and (C2) THP-1 cells transfected with either of the two different <i>CYP26B1</i> variants or empty vector. (D4) COS-1 cells transfected with different concentrations of the <i>CYP26B1</i> variants separately or in combination. The values are expressed as mean ± S.D., <i>n = 4</i>. Statistical significance between <i>CYP26B1</i> variants was analysed by Student’s t test and ANOVA, <i>*P<0.05,**P<0.01, ***P<0.001</i> (spliced variant vs. CYP26B1 full length).</p