![Protein expression of the CYP26B1 splice variant in HUVEC, AOSMCs and COS-1 cells. (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 CYP26B1 variants, both variants simultaneously, or empty vector after 24 hours treatment with either atRA or DMSO (control). (C1) Cellular concentration of exogenously added [ 3 H] atRA in COS-1 and (C2) THP-1 cells transfected with either of the two different CYP26B1 variants or empty vector. (D4) COS-1 cells transfected with different concentrations of the CYP26B1 variants separately or in combination. The values are expressed as mean 6 S.D., n = 4. Statistical significance between CYP26B1 variants was analysed by Student's t test and ANOVA, *P,0.05,**P,0.01, ***P,0.001 (spliced variant vs. CYP26B1 full length). doi:10.1371/journal.pone.0036839.g003](https://www.researchgate.net/profile/Ake-Strid/publication/225187220/figure/fig7/AS:669582205399045@1536652246298/Protein-expression-of-the-CYP26B1-splice-variant-in-HUVEC-AOSMCs-and-COS-1-cells-A1_Q320.jpg)
![Enzymatic and metabolic assay of CYP26B1. (A) CYP26B1 full length and splice variant were incubated with 25 nM and 100 nM of [ 3 H] atRA, respectively, for different time points as indicated. (B) CYP26B1 full length or splice variant was incubated with different concentrations of [ 3 H] atRA for 5 minutes and the disappearance of [ 3 H] atRA was followed. (C&D) CYP26B1 full length, splice variant, combination of both, and control, incubated with 100 nM of [ 3 H] atRA for 10 minutes(C) or incubated with 25nM of [ 3 H] atRA for different time points (D) as indicated in figure. The control used in the experiment was either full length CYP26B1 or spliced variant CYP26B1 without initiation by NADPH, the values in the figures are mean 6SD, n = 3 analyzed by Student's t-test (Fig4 A &B) and ANOVA (Fig 4C&D),*P,0.05, **P,0.01, ***P,0.001 vs. CYP26B1 full length. doi:10.1371/journal.pone.0036839.g004](https://www.researchgate.net/profile/Ake-Strid/publication/225187220/figure/fig8/AS:669582205407237@1536652246329/Enzymatic-and-metabolic-assay-of-CYP26B1-A-CYP26B1-full-length-and-splice-variant-were_Q320.jpg)
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Cloning and Functional Studies of a Splice Variant of CYP26B1 Expressed in Vascular Cells
PLOS ONE
Abstract and Figures
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.
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.
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.
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... A metabolic map of arecoline in mice revealed that CYP or a flavin-containing monooxygenase might create 3 forms of Noxide metabolites or arecoline [38]. Although the CYP26B1 splice variant lacks exon 2 in the coding region, it still has the ability to downgrade at-RA [39]. A previous study indicated that full-length CYP26B1 plays a crucial role in the catabolism of at-RA and regulation signaling [40], whereas the splice variant of CYP26B1 exerts slight or no influence on at-RA regulation [39]. ...
... Although the CYP26B1 splice variant lacks exon 2 in the coding region, it still has the ability to downgrade at-RA [39]. A previous study indicated that full-length CYP26B1 plays a crucial role in the catabolism of at-RA and regulation signaling [40], whereas the splice variant of CYP26B1 exerts slight or no influence on at-RA regulation [39]. ...
... Our arecoline cell toxicity data obtained from the MTT assay indicated that, when the quantity of CYP26B1 is higher, Ca9-22 cells are more susceptible to arecoline. This in vitro observation revealed that the expression of full-length CYP26B1 plays a greater role than does that of the CYP26B1 splice variant and is consistent with a previous report [39]. Using paired human oral cancer tissues with long-term chewing habits, we confirmed that the expression of the CYP26B1 splice variant was consistently higher in oral cancerous tissues than in adjacent noncancerous tissues. ...
Betel quid (BQ) is a psychostimulant, an addictive substance, and a group 1 carcinogen that exhibits the potential to induce adverse health effects. Approximately, 600 million users chew a variety of BQ. Areca nut (AN) is a necessary ingredient in BQ products. Arecoline is the primary alkaloid in the AN and can be metabolized through the cytochrome P450 (CYP) superfamily by inducing reactive oxygen species (ROS) production. Full-length CYP26B1 is related to the development of oral pharyngeal cancers. We investigated whether a splice variant of CYP26B1 is associated with the occurrence of ROS related oral and pharyngeal cancer. Cytotoxicity assays were used to measure the effects of arecoline on cell viability in a dose-dependent manner.
In vitro
and
in vivo
studies were conducted to evaluate the expression of the CYP26B1 splice variant. The CYP26B1 splice variant exhibited lower expression than did full-length CYP26B1 in the human gingival fibroblast-1 and Ca9-22 cell models. Increased expression of the CYP26B1 splice variant was observed in human oral cancer tissue compared with adjacent normal tissue, and increased expression was observed in patients at a late tumor stage. Our results suggested that the CYP26B1 splice variant is associated with the occurrence of BQ-related oral cancer.
... A polymorphic variation rs2241057C/C (minor allele) L264S is associated with larger macrophage-positive atherosclerotic lesions, whereas carries of the major allele rs2241057T/T have an increased risk of Crohn's disease, an autoimmune condition of the gut. Additionally, Cyp26b1 is upregulated in atheroschlerotic lesions and associated there with activated macrophages, suggesting that Cyp26b1 may be important in RA clearance in the arterial wall [215][216][217]. ...
This review focuses on the role of the Cytochrome p450 subfamily 26 (CYP26) retinoic acid (RA) degrading enzymes during development and regeneration. Cyp26 enzymes, along with retinoic acid synthesising enzymes, are absolutely required for RA homeostasis in these processes by regulating availability of RA for receptor binding and signalling. Cyp26 enzymes are necessary to generate RA gradients and to protect specific tissues from RA signalling. Disruption of RA homeostasis leads to a wide variety of embryonic defects affecting many tissues. Here, the function of CYP26 enzymes is discussed in the context of the RA signalling pathway, enzymatic structure and biochemistry, human genetic disease, and function in development and regeneration as elucidated from animal model studies.
... Several enzymes involved in retinoid metabolism and action are expressed in the vasculature including alcohol dehydrogenases and RARs (Allali-Hassani, Martinez, Peralba, Vaglenova, Vidal, Richart, et al., 1997;Miano, Topouzis, Majesky, & Olson, 1996), and RAR activity in human smooth muscle cells was confirmed in transient transfected reporter gene assays (Ocaya, Elmabsout, Olofsson, Torma, Gidlof, & Sirsjo, 2011). Of the three CYP26 enzymes, CYP26B1 was found to be constitutively expressed in human vascular smooth cells and play a major role in atRA clearance in the arterial wall (Elmabsout, Kumawat, Saenz-Méndez, Krivospitskaya, Sävenstrand, Olofsson, et al., 2012;Krivospitskaya, Elmabsout, Sundman, Soderstrom, Ovchinnikova, Gidlof, et al., 2012;Ocaya et al., 2011). Based on this, RA may also play a role in regulating other functions in the vasculature. ...
Vitamin A (retinol) and its active metabolite, all-trans-retinoic acid (atRA), play critical roles in regulating the differentiation, growth, and migration of immune cells. Similarly, as critical signaling molecules in the regulation of the cell cycle, retinoids are important in cancers. Concentrations of atRA are tightly regulated in tissues, predominantly by the availability of retinol, synthesis of atRA by ALDH1A enzymes and metabolism and clearance of atRA by CYP26 enzymes. The ALDH1A and CYP26 enzymes are expressed in several cell types in the immune system and in cancer cells. In the immune system, the ALDH1A and CYP26 enzymes appear to modulate RA concentrations. Consequently, alterations in the activity of ALDH1A and CYP26 enzymes are expected to change disease outcomes in inflammation. There is increasing evidence from various disease models of intestinal and skin inflammation that treatment with atRA has a positive effect on disease markers. However, whether aberrant atRA concentrations or atRA synthesis and metabolism play a role in inflammatory disease development and progression is not well understood. In cancers, especially in acute promyelocytic leukemia and neuroblastoma, increasing intracellular concentrations of atRA appears to provide clinical benefit. Inhibition of the CYP26 enzymes to increase atRA concentrations and combat therapy resistance has been pursued as a drug target in these cancers. This chapter covers the current knowledge of how atRA and retinol regulate the immune system and inflammation, how retinol and atRA metabolism is altered in inflammation and cancer, and what roles atRA-metabolizing enzymes have in immune responses and cancers.
... Cyp26b1 encodes protein Cytochrome P450 26B1, known to be present in adult mice skeletal muscle [85]. Cyp26b1 signals aortic smooth muscle cells through regulation of the metabolism of all-trans-retinoic acid, [86] which is crucial for regulation of gene expression, cell growth and differentiation [87]. Finally, the protein encoded by Tropomyosin alpha-3 (TPM3) is also essential for regulation of skeletal muscle contraction [88]. ...
Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current-controlling mechanosensitive ion channels. These important findings extend hypotheses of myostatin and physical activity master regulation of genes and gene pathways, impacting medical practices and therapies associated with muscle atrophy in humans and companion animal species and genome-enabled selection practices applied to food-production animal species.
... cigarette smoke. Oppositely, the most significantly down-regulated gene in vessels of PH-IPF versus PH-COPD from the retinol metabolism pathway was cytochrome P-450 (CYP) 26B1, which is expressed in endothelial, smooth muscle cells and in atherosclerotic lesions (35). Inhibition of CYP26B1 by profibrotic transforming growth factor-b has recently been reported (36). ...
Rationale: The development of pulmonary hypertension (PH) in patients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD) is associated with increased morbidity. Objectives: To elucidate whether vascular remodeling in a well characterized PH-COPD and PH-IPF patient cohort results from similar or divergent molecular changes. Methods: Vascular remodeling of donor, PH-COPD and PH-IPF pulmonary arteries was assessed. Laser capture microdissected (LCM) pulmonary artery profiles in combination with whole genome microarrays were performed. Measurements and Main results: Pulmonary arteries from COPD and IPF patients with PH exhibited remodeling of vascular layers and reduction of lumen area. Pathway analyses comparing normalized gene expression profiles obtained from PH-IPF or PH-COPD patients revealed the retinol and extracellular matrix (ECM) receptor interaction to be the most perturbed processes. Within the ECM-receptor pathway, differential regulation of five out of the top 10 results (collagen, type III, alpha 1 (COL3A1), tenascin C (TNC), collagen, type VI, alpha 3 (COL6A3), thrombospondin 2 (THBS2) and von Willebrand factor (vWF)) were verified by real-time PCR and immunohistochemical staining. Conclusions: Despite clinical and histological vascular remodeling in all PH-COPD and PH-IPF patients, differential gene expression pattern was present in pulmonary artery profiles. Several genes involved in retinol metabolism and ECM receptor interaction enable discrimination of vascular remodeling in PH-IPF or PH-COPD. This suggests that pulmonary arterial remodeling in PH-COPD and PH-IPF is caused by different molecular mechanisms and may require specific therapeutic options.
... However, unlike adherent stromal cultures and male gonads where Sertoli cells are the main constituent of the microenvironment, the adult bone marrow niche is a rather complex milieu (12). Interestingly, major components of the bone marrow microenvironment, osteoblasts (31) and endothelial cells (32), also have been shown to express CYP26. The process of physiological HSC self-renewal remains incompletely understood despite intense investigation. ...
Significance
Mechanisms that control physiologic hematopoietic stem-cell (HSC) self-renewal remain largely unknown. Inhibition of retinoic acid (RA) signaling in HSCs maintained their primitive phenotype and function, and promoted their self-renewal. Moreover, bone marrow stroma’s expression of the cytochrome P450 retinoid-inactivating enzyme, CYP26, allowed HSC self-renewal by maintaining an environment low in retinoids. Thus, HSCs appear to be intrinsically programmed to undergo RA-mediated differentiation unless prevented from doing so by bone marrow niche CYP26. Modulation of RA signaling also holds promise for clinical HSC expansion.
CYP26B1 is a member of the cytochrome P450 family and is responsible for the break-down of retinoic acid for which appropriate levels are important for normal development of the cardiovascular and lymphatic systems. In a cohort of 235 patients with lymphatic malformations, we performed genetic testing for the CYP26B1 gene. These probands had previously tested negative for known lymphedema genes. We identified two heterozygous missense CY-P26B1 variants in two patients. Our bioinformatic study suggested that alterations caused by these variants have no major effect on the overall stability of CYP26B1 protein structure. Balanced levels of retinoic acid maintained by CYP26B1 are crucial for the lymphatic system. We identified that CYP26B1 could be involved in predisposition for lymphedema. We propose that CYP26B1 be further explored as a new candidate gene for genetic testing of lymphedema patients.
The Cytochrome P450 (CYP) family 26 enzymes contribute to retinoic acid (RA) metabolism and homeostasis in humans, mammals and other chordates. The three CYP26 family enzymes, CYP26A1, CYP26B1 and CYP26C1 have all been shown to metabolize all-trans-retinoic acid (atRA) it's 9-cisRA and 13-cisRA isomers and primary metabolites 4-OH-RA and 4-oxo-RA with high efficiency. While no crystal structures of CYP26 enzymes are available, the binding of various ligands has been extensively explored via homology modeling. All three CYP26 enzymes are inducible by treatment with atRA in various prenatal and postnatal tissues and cell types. However, current literature shows that in addition to regulation by atRA, CYP26 enzyme expression is also regulated by other endogenous processes and inflammatory cytokines. In humans and in animal models the expression patterns of CYP26 enzymes have been shown to be tissue and cell type specific, and the expression of the CYP26 enzymes is believed to regulate the formation of critical atRA concentration gradients in various tissue types. Yet, very little data exists on direct disease associations of altered CYP26 expression or activity. Nevertheless, data is emerging describing a variety of human genetic variations in the CYP26 enzymes that are associated with different pathologies. Interestingly, some of these genetic variants result in increased activity of the CYP26 enzymes potentially leading to complex gene-environment interactions due to variability in dietary intake of retinoids. This review highlights the current knowledge of structure-function of CYP26 enzymes and focuses on their role in human retinoid metabolism in different tissues.
The human genome encodes 57 cytochrome P450 (CYP) genes whose enzyme products metabolize hundreds of drugs, thousands of xenobiotics and unknown numbers of endogenous compounds including steroids, retinoids and icosinoids. Indeed, CYP genes are the first line of defense against daily environmental chemical challenges in a manner that parallels the immune system. Several databases, including PubMed, AceView, and Ensembl, were queried to establish a comprehensive analysis of the full human CYP transcriptome. This review describes a remarkable diversification of the 57 human CYP genes, which may be alternatively processed into nearly 1000 distinct mRNA transcripts to shape an individual's CYP proteome. Important CYP splice variants from families: 1A, 1B, 2C, 2D, 3A, 4F, 19A and 24A have now been documented, with some displaying alternative subcellular distribution or catalytic function directly linked to a disease pathology. The expansion of CYP transcript diversity involves tissue-specific splicing factors, transformation-sensitive alternate splicing, trans-splicing between gene transcripts, single-nucleotide polymorphisms (SNPs), and epigenetic regulation of alternate splicing. Homeostatic regulation of variant CYP expression also is influenced by nuclear receptor (NR) signaling, suppression of nonsense mediated decay (NMD) or premature termination codons (PTC), mitochondrial dysfunction or host infection. This review focuses on emergent aspects of the adaptive gene splicing process, which when viewed through the lens of CYP-NR gene interactions, resembles a primitive immune-like system that can rapidly monitor, respond and diversify to acclimate to fluctuations in endo-xenobiotic exposure. Insights gained from this review should aid future drug discovery and improve therapeutic management of personalized drug regimens.
RNA-sequencing (RNA-seq) analysis enables to study gene-environment interactions and their impact on the transcriptome. The variety of techniques and software packages for statistical analysis available tailors RNA-seq processing for a specific experiment. Three individual studies, each dissecting a different environmental factor including exercise and a specific immunological challenge, resulted in significant findings. In the first study, mice with the indoleamine 2,3-dioxygenase gene knocked out suffered from the same inflammation as wild type mice when injected with Bacillus Calmette-Guerin, but interestingly did not exhibit any of the depressive symptomatology which characteristically accompanies the infection. RNA-seq data was extracted from these mice, and statistical analysis revealed which genes may be responsible for this phenomenon, which could potentially provide researchers with the tools to create genetic treatments of depression occurring as a byproduct of inflammatory disorders. The second study of gene factors in Myostatin knock-out mice that were either allowed to or restricted from exercise revealed novel gene links for muscle development. Knowledge of genes involved in muscle growth post-development can allow for treatment of muscle atrophy in adults. A final study aimed to use a dependence-like preference for exercise as a murine model for substance addiction. Mice were selected for their drive towards exercise and then either allowed or deprived of the reward. These results showcase the power of advanced statistical methods of RNA-seq analysis. Copyright © (2014) by the International Society for Computers and Their Applications.
All-trans retinoic acid, controlled by cytochrome P450, family 26 (CYP26) enzymes, potentially has beneficial effects in atherosclerosis treatment. This study investigates CYP26 subfamily B, polypeptide 1 (CYP26B1) in atherosclerosis and the effects of a genetic polymorphism in CYP26B1 on retinoid catabolism. We found that CYP26B1 mRNA was induced by retinoic acid in human atherosclerotic arteries, and CYP26B1 and the macrophage marker CD68 were colocalized in human atherosclerotic lesions. In mice, Cyp26B1 mRNA was higher in atherosclerotic arteries than in normal arteries. Databases were queried for nonsynonymous CYP26B1 single nucleotide polymorphisms (SNPs) and rs2241057 selected for further studies. Constructs of the CYP26B1 variants were created and used for production of purified proteins and transfection of macrophagelike cells. The minor variant catabolized retinoic acid with significantly higher efficiency, indicating that rs2241057 is functional and suggesting reduced retinoid availability in tissues with the minor variant. rs2241057 was investigated in a Stockholm Coronary Atherosclerosis Risk Factor (SCARF) subgroup. The minor allele was associated with slightly larger lesions, as determined by angiography. In summary, this study identifies the first CYP26B1 polymorphism that alters CYP26B1 capacity to metabolize retinoic acid. CYP26B1 was expressed in macrophage-rich areas of human atherosclerotic lesions, induced by retinoic acid and increased in murine atherosclerosis. Taken together, the results indicate that CYP26B1 capacity is genetically regulated and suggest that local CYP26B1 activity may influence atherosclerosis.
Retinoic acid (RA) regulates the differentiation and proliferation of a wide variety of different cell types and all-trans RA induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). However, clinical resistance to retinoids may develop and poses a serious problem for differentiation-inducing therapy. We studied the effects of RA in combination with a cytochrome P450 inhibitor (clotrimazole) and a P-glycoprotein antagonist (verapamil) on cell growth and differentiation of RA-resistant HL-60 cells and fresh RA-resistant leukemic cells from two APL patients. RA-resistant HL-60 cells and APL cells differentiated to mature granulocytes when cultured with all-trans RA and either clotrimazole and verapamil but not with either of the agents alone. These findings were confirmed in these cells by their increased expression of CD11b antigen and migration-inhibitory factor-related protein-8/14 mRNAs and decreased levels of c-myc mRNA. These combinations also markedly decreased the number of viable cells and inhibited cellular proliferation. After isolation of microsomes, measurements showed that levels of cytochrome P450 activities in both wild-type and RA-resistant HL-60 cells were almost comparable. Moreover, expression of the CYP1A1-type cytochrome P450 gene could not be detected in either cell type. However, RA-resistant HL-60 cells and APL cells, but not RA-sensitive HL-60 cells and APL cells, expressed multidrug-resistance-1 gene transcripts. Taken together, acquired resistance to RA may be explained in part by drug metabolism in leukemic cells. Possible mechanisms for accelerated clearance of RA include the induction of non-CYP1A1 cytochrome P450 enzymes and P-glycoprotein.
Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all-trans RA into 5,8-epoxy all-trans RA. P450RA metabolizes other biologically active RAs such as 9-cis RA and 13-cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all-trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage- and region-specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.
Retinoids, the metabolically-active structural derivatives of vitamin A, are critical signaling molecules in many fundamental biological processes including cell survival, proliferation and differentiation. Emerging evidence, both clinical and molecular, implicates retinoids in atherosclerosis and other vasculoproliferative disorders such as restenosis. Although the data from clinical trials examining effect of vitamin A and vitamin precursors on cardiac events have been contradictory, this data does suggest that retinoids do influence fundamental processes relevant to atherosclerosis. Preclinical animal model and cellular studies support these concepts. Retinoids exhibit complex effects on proliferation, growth, differentiation and migration of vascular smooth muscle cells (VSMC), including responses to injury and atherosclerosis. Retinoids also appear to exert important inhibitory effects on thrombosis and inflammatory responses relevant to atherogenesis. Recent studies suggest retinoids may also be involved in vascular calcification and endothelial function, for example, by modulating nitric oxide pathways. In addition, established retinoid effects on lipid metabolism and adipogenesis may indirectly influence inflammation and atherosclerosis. Collectively, these observations underscore the scope and complexity of retinoid effects relevant to vascular disease. Additional studies are needed to elucidate how context and metabolite-specific retinoid effects affect atherosclerosis. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.
The active metabolite of vitamin A, retinoic acid (RA), is a powerful regulator of gene transcription. RA is also a therapeutic drug. The oxidative metabolism of RA by certain members of the cytochrome P450 (CYP) superfamily helps to maintain tissue RA concentrations within appropriate bounds. The CYP26 family--CYP26A1, CYP26B1, and CYP26C1--is distinguished by being both regulated by and active toward all-trans-RA (at-RA) while being expressed in different tissue-specific patterns. The CYP26A1 gene is regulated by multiple RA response elements. CYP26A1 is essential for embryonic development, whereas CYP26B1 is essential for postnatal survival as well as germ cell development. Enzyme kinetic studies have demonstrated that several CYP proteins are capable of metabolizing at-RA; however, it is likely that CYP26A1 plays a major role in RA clearance. Thus, pharmacological approaches to limiting the activity of CYP26 enzymes may extend the half-life of RA and could be useful clinically in the future.
The cytochrome P450 enzymes of the CYP26 family are involved in the catabolism of the biologically active retinoid all-trans-retinoic acid (atRA). Since it is possible that an increased local CYP26 activity would reduce the effects of retinoids in vascular injury, we investigated the role of CYP26 in the regulation of atRA levels in human aortic smooth muscle cells (AOSMCs).
The expression of CYP26 was investigated in cultured AOSMCs using real-time PCR. The metabolism of atRA was analyzed by high-performance liquid chromatography, and the inhibitor R115866 or small interfering RNA (siRNA) was used to suppress CYP26 activity/expression.
AOSMCs expressed CYP26B1 constitutively and atRA exposure augmented CYP26B1 mRNA levels. Silencing of the CYP26B1 gene expression or reduction of CYP26B1 enzymatic activity by using siRNA or the inhibitor R115866, respectively, increased atRA-mediated signaling and resulted in decreased cell proliferation. The CYP26 inhibitor also induced expression of atRA-responsive genes. Therefore, atRA-induced CYP26 expression accelerated atRA inactivation in AOSMCs, giving rise to an atRA-CYP26 feedback loop. Inhibition of this loop with a CYP26 inhibitor increased retinoid signaling.
The results suggest that CYP26 inhibitors may be a therapeutic alternative to exogenous retinoid administration.
All-trans retinoic acid (RA) levels are controlled by enzymes of the vitamin A metabolism (RDH16, RalDH2, and LRAT) and RA catabolism (CYP26 and CYP2S1). Here, the mRNA expression of these enzymes was investigated in human keratinocytes at different Ca(2+)concentrations and after exposure to RA and CYP26 inhibitors. Cellular differentiation (high Ca(2+)) increased the expression of LRAT, RDH16 and RalDH2, and decreased CYP26B1. RA (1 microM) induced CYP26A1, CYP26B1, CYP2S1, CRABPII and LRAT mRNA. The CYP26 inhibitor talarozole altered CYP26A1 and LRAT mRNA expression in a similar way as RA, increased the cellular accumulation of [(3)H]RA, and induced a punctate CRABPII staining, also observed after siRNA knock-down of CYP26B1 (but not after RA exposure). Furthermore, CYP26B1 siRNA increased the accumulation of [(3)H]RA and the CRABPII mRNA, suggesting an augmented retinoid signalling. Thus CYP26B1 appears essential for RA catabolism under physiological conditions, whereas CYP26A1 might play a greater role during RA excess.
An alternative approach to retinoid therapy is to inhibit the cytochrome P450 (CYP)-mediated catabolism of endogenous all-trans retinoic acid in the skin by applying retinoic acid metabolism blocking agents such as talarozole (R115866).
To study the effects of topical talarozole on retinoid biomarkers in normal skin in a randomized phase I trial.
Gels containing talarozole (0.35% or 0.07%) and vehicle were applied once daily for 9 days on either buttock of 16 healthy volunteers. Epidermal shave biopsies (for mRNA analysis) and punch biopsies (for histology and immunofluorescence analysis) were collected from the treatment areas. Genes encoding the following were studied by quantitative real-time polymerase chain reaction: cellular retinoic acid binding protein 2 (CRABP2), cytokeratins (KRT2 and KRT4), CYP26A1, CYP26B1, CYP26C1 and CYP2S1, two enzymes in the retinol metabolism (retinal dehydrogenase-2 and retinol acyltransferase) and two proinflammatory cytokines [interleukin (IL)-1alpha and tumour necrosis factor-alpha].
Talarozole treatment increased the mRNA expression of CRABP2, KRT4, CYP26A1 and CYP26B1 dose dependently, and decreased the expression of KRT2 and IL-1alpha compared with vehicle-treated skin. No mRNA change in retinol-metabolizing enzymes was obtained. There was no induction of epidermal thickness or overt skin inflammation in talarozole-treated skin. Immunofluorescence analysis confirmed an upregulation of KRT4 protein, but no upregulation of CYP26A1 and CYP26B1 expression was detected.
Talarozole influences the biomarker pattern consistently with increased retinoic acid stimulation. The low irritancy of talarozole at the two examined dosages is a possible advantage over topical retinoids.
In two independent human cohorts, the minor allele of SNP rs3850641 in TNFSF4 was significantly more frequent in individuals with myocardial infarction than in controls. In mice, Tnfsf4 expression is associated with increased atherosclerosis. The expression of TNFSF4 in human atherosclerosis and the association between genotype and cerebrovascular disease have not yet been investigated. TNFSF4 messenger RNA (mRNA) levels were significantly higher in human atherosclerotic lesions compared with controls (730 +/- 30 vs 330 +/- 65 arbitrary units, p < 0.01). TNFSF4 was mainly expressed by macrophages in atherosclerotic lesions. In cell culture, endothelial cells upregulated TNFSF4 in response to tumor necrosis factor alpha (TNF-alpha; 460 +/- 110 vs 133 +/- 8 arbitrary units, p < 0.001 after 6 h of stimulation). We analyzed the TNFSF4 gene in 239 patients who had undergone carotid endarterectomy and 138 matching controls from The Biobank of Karolinska Carotid Endarterectomies and Stockholm Heart Epidemiology Program cohorts and 929 patients and 1,382 matching controls from the Sahlgrenska Academy Study on Ischemic Stroke and Case Control Study of Stroke cohorts, limiting inclusion to patients with ischemic stroke. Participants were genotyped for the rs3850641 SNP in TNFSF4. Genotype associations were neither found with TNFSF4 mRNA levels nor with atherosclerosis associated systemic factors or risk for stroke. This study shows that TNFSF4 is expressed on antigen-presenting cells in human carotid atherosclerotic lesions but provides no evidence for an association of TNFSF4 gene variation with the risk for ischemic stroke.
We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.