Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome

BACKGROUND: The retinoic acid (RA)-catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1-null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS: Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by chi(2) analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS: Mutational screening allowed us to identify 6 SNPs, 4 of which (447 C>G, 1134 G>A, IVS 1+10 G>C, and IVS 4+8 AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS: Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects.     

Expression of the retinoic acid catabolising enzyme CYP26B1 in the chick embryo and its regulation by retinoic acid

We have cloned a fragment of Cyp26B1, a novel retinoic acid (RA) catabolising enzyme, and examined its expression pattern during early stages of chick embryogenesis. It is expressed from stage 7 in the tail bud, an anterior patch of mesenchyme, the heart, the endothelium of the vasculature, the eye, the limb bud, the hindgut and in a complex pattern in the rhombomeres of the hindbrain. As such it has a non-overlapping expression with chick Cyp26A1, the other RA catabolising enzyme, but shows a combination of features of mouse Cyp26A1 and Cyp26B1. We have also examined its expression in the quail embryo and in the RA-free quail embryo. In the absence of RA, Cyp26B1 is only expressed in the hindbrain and fails to be expressed in all the other regions of the embryo, most dramatically in the trunk. Adding back RA rescues Cyp26B1 expression.     

Growth differentiation factor 11 signaling controls retinoic acid activity for axial vertebral development

Mice deficient in growth differentiation factor 11 (GDF11) signaling display anterior transformation of axial vertebrae and truncation of caudal vertebrae. However, the in vivo molecular mechanisms by which GDF11 signaling regulates the development of the vertebral column have yet to be determined. We found that Gdf11 and Acvr2b mutants are sensitive to exogenous RA treatment on vertebral specification and caudal vertebral development. We show that diminished expression of Cyp26a1, a retinoic acid inactivating enzyme, and concomitant elevation of retinoic acid activity in the caudal region of Gdf11^−/− embryos may account for this phenomenon. Reduced expression or function of Cyp26a1 enhanced anterior transformation of axial vertebrae in wild-type and Acvr2b mutants. Furthermore, a pan retinoic acid receptor antagonist (AGN193109) could lessen the anterior transformation phenotype and rescue the tail truncation phenotype of Gdf11^−/− mice. Taken together, these results suggest that GDF11 signaling regulates development of caudal vertebrae and is involved in specification of axial vertebrae in part by maintaining Cyp26a1 expression, which represses retinoic acid activity in the caudal region of embryos during the somitogenesis stage.     

Regulation of retinoic acid distribution is required for proximodistal patterning and outgrowth of the developing mouse limb

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.     

The control of morphogen signalling: regulation of the synthesis and catabolism of retinoic acid in the developing embryo

We consider here how morphogenetic signals involving retinoic acid (RA) are switched on and off in the light of positive and negative feedback controls which operate in other embryonic signalling systems. Switching on the RA signal involves the synthetic retinaldehyde dehydrogenase (RALDH) enzymes and it is currently thought that switching off the RA signal involves the CYP26 enzymes which catabolise RA. We have tested whether these enzymes are regulated by the presence or absence of all-trans-RA using the vitamin A-deficient quail model system and the application of excess retinoids on beads to various locations within the embryo. The Raldhs are unaffected either by the absence or presence of excess RA, whereas the Cyps are strongly affected. In the absence of RA some, but not all domains of Cyp26A1, Cyp26B1 and Cyp26C1 are down-regulated, in particular the spinal cord (Cyp26A1), the heart and developing vasculature (Cyp26B1) and the rhombomeres (Cyp26C1). In the presence of excess RA, the Cyps show a differential regulation-Cyp26A1 and Cyp26B1 are up-regulated whereas Cyp26C1 is down-regulated. We tested whether the Cyp products have a similar influence on these genes and indeed 4-oxo-RA, 4-OH-RA and 5,6-epoxy-RA do. Furthermore, these 3 metabolites are biologically active in that they fully rescue the vitamin A-deficient quail embryo. Finally, by using retinoic acid receptor selective agonists we show that these compounds regulate the Cyps through the RARalpha receptor. These results are discussed with regard to positive and negative feedback controls in developing systems.     

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis

Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.     

Analysis of Cyp26b1/Rarg compound-null mice reveals two genetically separable effects of retinoic acid on limb outgrowth

The role of retinoic acid (RA) in limb development is unclear, although it has been suggested to be a proximalizing factor which plays a morphogenetic role in pattern formation. Exogenous RA produces a teratogenic effect on limb morphology; similarly, changes in the endogenous distribution of RA following genetic ablation of the RA-metabolizing enzyme, CYP26B1, result in phocomelia accompanied by changes in expression of proximo-distal (P-D) patterning genes, increased cell death, and delayed chondrocyte maturation. Here we show that disruption of RA receptor (RAR) gamma in a Cyp26b1^−/− background is able to partially rescue limb skeletal morphology without restoring normal expression of proximo-distal patterning genes. We further show that embryos deficient in CYP26B1 exhibit early localized domains of mesenchymal cell death, which are reduced in compound-null animals. This model reveals two genetically separable effects of RA in the limb: an apoptotic effect mediated by RARγ in the presence of ectopic RA, and a P-D patterning defect which is uncovered following the loss of both CYP26B1 and RARγ. These data provide genetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distal patterning.     

CYP26B1 promotes male germ cell differentiation by suppressing STRA8-dependent meiotic and STRA8-independent mitotic pathways

Germ cell sex is defined by factors derived from somatic cells. CYP26B1 is known to be a male sex-promoting factor that inactivates retinoic acid (RA) in somatic cells. In CYP26B1-null XY gonads, germ cells are exposed to a higher level of RA than in normal XY gonads and this activates Stra8 to induce meiosis while male-specific gene expression is suppressed. However, it is unknown whether meiotic entry by an elevated level of RA is responsible for the suppression of male-type gene expression. To address this question, we have generated Cyp26b1/Stra8 double knockout (dKO) embryos. We successfully suppressed the induction of meiosis in CYP26B1-null XY germ cells by removing the Stra8 gene. Concomitantly, we found that the male genetic program represented by the expression of NANOS2 and DNMT3L was totally rescued in about half of dKO germ cells, indicating that meiotic entry causes the suppression of male differentiation. However, half of the germ cells still failed to enter the appropriate male pathway in the dKO condition. Using microarray analyses together with immunohistochemistry, we found that KIT expression was accompanied by mitotic activation, but was canceled by inhibition of the RA signaling pathway. Taken together, we conclude that inhibition of RA is one of the essential factors to promote male germ cell differentiation, and that CYP26B1 suppresses two distinct genetic programs induced by RA: a Stra8-dependent meiotic pathway, and a Stra8-independent mitotic pathway.     

Retinoid regulated association of transcriptional co-regulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARbeta(2), and Cyp26A1 in F9 embryonal carcinoma cells

Hox gene expression is activated by all-trans retinoic acid (RA), through binding to retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (alpha, beta, and gamma), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRalpha, RARgamma, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARbeta2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARgamma and RXRalpha are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of co-regulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.     

CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse

The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1-/-Cyp26c1-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.     

Inhibition of Tgf beta signaling by endogenous retinoic acid is essential for primary lung bud induction

Disruption of retinoic acid (RA) signaling during early development results in severe respiratory tract abnormalities, including lung agenesis. Previous studies suggest that this might result from failure to selectively induce fibroblast growth factor 10 (Fgf10) in the prospective lung region of the foregut. Little is known about the RA-dependent pathways present in the foregut that may be crucial for lung formation. By performing global gene expression analysis of RA-deficient foreguts from a genetic [retinaldehyde dehydrogenase 2 (Raldh2)-null] and a pharmacological (BMS493-treated) mouse model, we found upregulation of a large number of Tgfbeta targets. Increased Smad2 phosphorylation further suggested that Tgfbeta signaling was hyperactive in these foreguts when lung agenesis was observed. RA rescue of the lung phenotype was associated with low levels of Smad2 phosphorylation and downregulation of Tgfbeta targets in Raldh2-null foreguts. Interestingly, the lung defect that resulted from RA-deficiency could be reproduced in RA-sufficient foreguts by hyperactivating Tgfbeta signaling with exogenous TGF beta 1. Preventing activation of endogenous Tgfbeta signaling with a pan-specific TGFbeta-blocking antibody allowed bud formation and gene expression in the lung field of both Raldh2-null and BMS493-treated foreguts. Our data support a novel mechanism of RA-Tgfbeta-Fgf10 interactions in the developing foregut, in which endogenous RA controls Tgfbeta activity in the prospective lung field to allow local expression of Fgf10 and induction of lung buds.     

Retinoids, eye development, and maturation of visual function

Vitamin A is known to be critical for the beginning of eye development as well as for photoreception in the functional retina. Hardly anything, however, is known about whether retinoic acid (RA)-regulated gene expression also plays a role in the long intervening period, during which the neurobiological retinal structure takes shape. The eye contains a highly intricate architecture of RA-synthesizing (RALDH) and degrading (CYP26) enzymes. Whereas the RALDHs are integrated in the early molecular mechanisms through which the dorso-ventral retina organization is established, the CYP26 enzymes are not necessary for this process and no molecular targets that match their retinal expression pattern have yet been identified. In this article we describe that CYP26 expression in the mouse is most distinctive during later stages of retina formation. Throughout development CYP26A1 degrades RA in a horizontal region that extends across the retina, but during later embryonic and postnatal retina maturation this function is reinforced by another enzyme, CYP26C1. RA applications at this stage do not affect the RALDHs but cause differential changes in CYP26 expression: Cyp26a1 is up-regulated, but more rapidly by 9-cis than all-trans RA, Cyp26c1 is down-regulated, and Cyp26b1, which is undetectable in the normal mouse retina, is strongly activated in retinal ganglion cells. The dynamic regulation in RA-difference patterns by the CYP26 enzymes may set up spatial constellations for expression of genes involved in formation of retinal specializations for higher acuity vision, which are known to form over a prolonged period late in retina development.     

Stage-dependent responses of the developing lung to retinoic acid signaling

Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis.     

A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of all-trans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA.     

Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors

In the embryonic mouse retina, retinoic acid (RA) is unevenly distributed along the dorsoventral axis: RA-rich zones in dorsal and ventral retina are separated by a horizontal RA-poor stripe that contains the RA-inactivating enzyme CYP26A1. To explore the developmental role of this arrangement, we studied formation of the retina and its projections in Cyp26a1 null-mutant mice. Expression of several dorsoventral markers was not affected, indicating that CYP26A1 is not required for establishing the dorsoventral retina axis. Analysis of the mutation on a RA-reporter mouse background confirmed, as expected, that the RA-poor stripe was missing in the retina and its projections at the time when the optic axons first grow over the diencephalon. A day later, however, a gap appeared both in retina and retinofugal projections. As explanation, we found that CYP26C1, another RA-degrading enzyme, had emerged centrally in a narrower domain within the RA-poor stripe. While RA applications increased retinal Cyp26a1 expression, they slightly reduced Cyp26c1. These observations indicate that the two enzymes function independently. The safeguard of the RA-poor stripe by two distinct enzymes during later development points to a role in maturation of a significant functional feature like an area of higher visual acuity that develops at its location.     

Cyp26b1 regulates retinoic acid-dependent signals in T cells and its expression is inhibited by transforming growth factor-β

Background

The vitamin A metabolite, retinoic acid (RA), plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF)-β-dependent induction of Foxp3⁺ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA.

Methodology/Principal Findings

We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44⁺ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1–10 nM) could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression.

Conclusions/Significance

Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3⁺ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs.