Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data.

Mixtures of substances sharing the same molecular initiating event (MIE) are supposed to induce additive effects. The proposed MIE for azole fungicides is CYP26 inhibition with retinoic acid (RA) local increase, triggering key events leading to craniofacial defects. Valproic acid (VPA) is supposed to imbalance RA-regulated gene expression trough histone deacetylases (HDACs) inhibition. The aim was to evaluate effects of molecules sharing the same MIE (azoles) and of such having (hypothetically) different MIEs but which are eventually involved in the same adverse outcome pathway (AOP). An in silico approach (molecular docking) investigated the suggested MIEs. Teratogenicity was evaluated in vitro (WEC). Abnormalities were modelled by PROAST software. The common target was the branchial apparatus. In silico results confirmed azole-related CYP26 inhibition and a weak general VPA inhibition on the tested HDACs. Unexpectedly, VPA showed also a weak, but not marginal, capability to enter the CYP 26A1 and CYP 26C1 catalytic sites, suggesting a possible role of VPA in decreasing RA catabolism, acting as an additional MIE. Our findings suggest a new more complex picture. Consequently two different AOPs, leading to the same AO, can be described. VPA MIEs (HDAC and CYP26 inhibition) impinge on the two converging AOPs.

Biochemical and physiological importance of the CYP26 retinoic acid hydroxylases.

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.

Highly Sensitive Quantitative Determination of Retinoic Acid Levels, Retinoic Acid Synthesis, and Catabolism in Embryonic Tissue Using a Reporter Cell-Based Method.

The effect of all-trans retinoic acid (RA) on embryogenesis is tissue specific and highly concentration dependent. Using a liquid chromatography/mass spectrometry-based method to quantify trace amounts of RA in embryonic tissue requires expensive specialist facilities. Here, we describe the use of a RA response element (RARE)-lacZ reporter cell-based method, which is simple and cost effective, to measure RA levels in small pieces of tissue from the embryo. We further apply this method to quantitatively assay activities of RA-synthesizing and RA-catabolizing enzymes, the key regulators of RA bioavailability in tissues and developing organs of the embryo.

Differential RA responsiveness directs formation of functionally distinct spermatogonial populations at the initiation of spermatogenesis in the mouse.

In the mammalian testis, sustained spermatogenesis relies on spermatogonial stem cells (SSCs); their progeny either remain as stem cells (self-renewal) or proliferate and differentiate to enter meiosis in response to retinoic acid (RA). Here, we sought to uncover elusive mechanisms regulating a key switch fundamental to spermatogonial fate: the capacity of spermatogonia to respond to RA. Using the developing mouse testis as a model, we found that spermatogonia and precursor prospermatogonia exhibit a heterogeneous capacity to respond to RA with at least two underlying causes. First, progenitor spermatogonia are prevented from responding to RA by catabolic activity of cytochrome P450 family 26 enzymes. Second, a smaller subset of undifferentiated spermatogonia enriched for SSCs exhibit catabolism-independent RA insensitivity. Moreover, for the first time, we observed that precursor prospermatogonia are heterogeneous and comprise subpopulations that exhibit the same differential RA responsiveness found in neonatal spermatogonia. We propose a novel model by which mammalian prospermatogonial and spermatogonial fates are regulated by their intrinsic capacity to respond (or not) to the differentiation signal provided by RA before, and concurrent with, the initiation of spermatogenesis.

Functional missense and splicing variants in the retinoic acid catabolizing enzyme CYP26C1 in idiopathic short stature.

Height is a complex quantitative trait with a high heritability. Short stature is diagnosed when height is significantly below the average of the general population for that person's age and sex. We have recently found that the retinoic acid degrading enzyme CYP26C1 modifies SHOX deficiency phenotypes toward more severe clinical manifestations. Here, we asked whether damaging variants in CYP26C1 alone could lead to short stature. We performed exome and Sanger sequencing to analyze 856 individuals with short stature where SHOX deficiency was previously excluded. Three different damaging missense variants and one splicing variant were identified in six independent individuals; the functional significance of the identified variants was tested in vitro or in vivo using zebrafish as a model. The genetic and functional data reported here indicate that CYP26C1 represents a novel gene underlying growth disorders and that damaging variants in the absence of SHOX variants can lead to short stature.

CYP26C1 Is a Hydroxylase of Multiple Active Retinoids and Interacts with Cellular Retinoic Acid Binding Proteins.

The clearance of retinoic acid (RA) and its metabolites is believed to be regulated by the CYP26 enzymes, but the specific roles of CYP26A1, CYP26B1, and CYP26C1 in clearing active vitamin A metabolites have not been defined. The goal of this study was to establish the substrate specificity of CYP26C1, and determine whether CYP26C1 interacts with cellular retinoic acid binding proteins (CRABPs). CYP26C1 was found to effectively metabolize all-trans retinoic acid (atRA), 9-cis-retinoic acid (9-cis-RA), 13-cis-retinoic acid, and 4-oxo-atRA with the highest intrinsic clearance toward 9-cis-RA. In comparison with CYP26A1 and CYP26B1, CYP26C1 resulted in a different metabolite profile for retinoids, suggesting differences in the active-site structure of CYP26C1 compared with other CYP26s. Homology modeling of CYP26C1 suggested that this is attributable to the distinct binding orientation of retinoids within the CYP26C1 active site. In comparison with other CYP26 family members, CYP26C1 was up to 10-fold more efficient in clearing 4-oxo-atRA (intrinsic clearance 153 µl/min/pmol) than CYP26A1 and CYP26B1, suggesting that CYP26C1 may be important in clearing this active retinoid. In support of this, CRABPs delivered 4-oxo-atRA and atRA for metabolism by CYP26C1. Despite the tight binding of 4-oxo-atRA and atRA with CRABPs, the apparent Michaelis-Menten constant in biological matrix (Km) value of these substrates with CYP26C1 was not increased when the substrates were bound with CRABPs, in contrast to what is predicted by free drug hypothesis. Together these findings suggest that CYP26C1 is a 4-oxo-atRA hydroxylase and may be important in regulating the concentrations of this active retinoid in human tissues.

Generating retinoic acid gradients by local degradation during craniofacial development: One cell's cue is another cell's poison.

Retinoic acid (RA) is a vital morphogen for early patterning and organogenesis in the developing embryo. RA is a diffusible, lipophilic molecule that signals via nuclear RA receptor heterodimeric units that regulate gene expression by interacting with RA response elements in promoters of a significant number of genes. For precise RA signaling, a robust gradient of the morphogen is required. The developing embryo contains regions that produce RA, and specific intracellular concentrations of RA are created through local degradation mediated by Cyp26 enzymes. In order to elucidate the mechanisms by which RA executes precise developmental programs, the kinetics of RA metabolism must be clearly understood. Recent advances in techniques for endogenous RA detection and quantification have paved the way for mechanistic studies to shed light on downstream gene expression regulation coordinated by RA. It is increasingly coming to light that RA signaling operates not only at precise concentrations but also employs mechanisms of degradation and feedback inhibition to self-regulate its levels. A global gradient of RA throughout the embryo is often found concurrently with several local gradients, created by juxtaposed domains of RA synthesis and degradation. The existence of such local gradients has been found especially critical for the proper development of craniofacial structures that arise from the neural crest and the cranial placode populations. In this review, we summarize the current understanding of how local gradients of RA are established in the embryo and their impact on craniofacial development.

Structural and functional characterisation of the cytochrome P450 enzyme CYP268A2 from Mycobacterium marinum.

Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of Mycobacterium species including the pathogens Mycobacterium avium, M. colombiense, M. kansasii, and Mmarinum CYP268A2, from M. marinum, which is the first member of this family to be studied, was purified and characterised. CYP268A2 was found to bind a variety of substrates with high affinity, including branched and straight chain fatty acids (C10-C12), acetate esters, and aromatic compounds. The enzyme was also found to bind phenylimidazole inhibitors but not larger azoles, such as ketoconazole. The monooxygenase activity of CYP268A2 was efficiently reconstituted using heterologous electron transfer partner proteins. CYP268A2 hydroxylated geranyl acetate and trans-pseudoionone at a terminal methyl group to yield (2E,6E)-8-hydroxy-3,7-dimethylocta-2,6-dien-1-yl acetate and (3E,5E,9E)-11-hydroxy-6,10-dimethylundeca-3,5,9-trien-2-one, respectively. The X-ray crystal structure of CYP268A2 was solved to a resolution of 2.0 Šwith trans-pseudoionone bound in the active site. The overall structure was similar to that of the related phytanic acid monooxygenase CYP124A1 enzyme from Mycobacterium tuberculosis, which shares 41% sequence identity. The active site is predominantly hydrophobic, but includes the Ser99 and Gln209 residues which form hydrogen bonds with the terminal carbonyl group of the pseudoionone. The structure provided an explanation on why CYP268A2 shows a preference for shorter substrates over the longer chain fatty acids which bind to CYP124A1 and the selective nature of the catalysed monooxygenase activity.

Retinoic acid, CYP26, and drug resistance in the stem cell niche.

The bone marrow niche is essential for hematopoietic stem cells to maintain lifelong blood production by balancing their self-renewal and differentiation. Hematologic malignancies have a similar hierarchical organization to their normal counterparts, with rare populations of cancer stem cells that rely on the microenvironment to survive and propagate their differentiated malignant progenitor cells. Cancer cells alter their microenvironment to create a supportive niche, where they endure chemotherapy, survive as minimal residual disease (MRD), and eventually prevail at relapse. Powerful morphogens, such as retinoids, Wnt/ßcatenin, Notch, and Hedgehog, control stem cell fates across tissues, including normal and malignant hematopoiesis. The molecular conversations between these pathways and the mechanisms that control their activity and create gradients at cellular scale remain a mystery. Here, we discuss accumulating evidence suggesting that cytochrome P450 (CYP26), the primary retinoid-inactivating enzyme, plays a critical role in the integration of two of these molecular programs: the retinoid and Hedgehog pathways. Induction of stromal CYP26 by either one of these pathways limits retinoic acid concentration in the stem cell niche, with profound effects on tissue homeostasis and drug resistance. Bypassing this gatekeeping mechanism holds promise for overcoming drug resistance and improving clinical outcomes in hematological malignancies and cancer in general.

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid.

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Lineage-specific duplication of amphioxus retinoic acid degrading enzymes (CYP26) resulted in sub-functionalization of patterning and homeostatic roles.

BACKGROUND: During embryogenesis, tight regulation of retinoic acid (RA) availability is fundamental for normal development. In parallel to RA synthesis, a negative feedback loop controlled by RA catabolizing enzymes of the cytochrome P450 subfamily 26 (CYP26) is crucial. In vertebrates, the functions of the three CYP26 enzymes (CYP26A1, CYP26B1, and CYP26C1) have been well characterized. By contrast, outside vertebrates, little is known about CYP26 complements and their biological roles. In an effort to characterize the evolutionary diversification of RA catabolism, we studied the CYP26 genes of the cephalochordate amphioxus (Branchiostoma lanceolatum), a basal chordate with a vertebrate-like genome that has not undergone the massive, large-scale duplications of vertebrates. RESULTS: In the present study, we found that amphioxus also possess three CYP26 genes (CYP26-1, CYP26-2, and CYP26-3) that are clustered in the genome and originated by lineage-specific duplication. The amphioxus CYP26 cluster thus represents a useful model to assess adaptive evolutionary changes of the RA signaling system following gene duplication. The characterization of amphioxus CYP26 expression, function, and regulation by RA signaling demonstrated that, despite the independent origins of CYP26 duplicates in amphioxus and vertebrates, they convergently assume two main roles during development: RA-dependent patterning and protection against fluctuations of RA levels. Our analysis suggested that in amphioxus RA-dependent patterning is sustained by CYP26-2, while RA homeostasis is mediated by CYP26-1 and CYP26-3. Furthermore, comparisons of the regulatory regions of CYP26 genes of different bilaterian animals indicated that a CYP26-driven negative feedback system was present in the last common ancestor of deuterostomes, but not in that of bilaterians. CONCLUSIONS: Altogether, this work reveals the evolutionary origins of the RA-dependent regulation of CYP26 genes and highlights convergent functions for CYP26 enzymes that originated by independent duplication events, hence establishing a novel selective mechanism for the genomic retention of gene duplicates.

Retinoic Acid Synthesis and Degradation.

Retinoic acid (RA) was identified as the biologically active form of vitamin A almost 70 years ago and work on its function and mechanism of action is still of major interest both from a scientific and a clinical perspective. The currently accepted model postulates that RA is produced in two sequential oxidative steps: first, retinol is oxidized reversibly to retinaldehyde, and then retinaldehyde is oxidized irreversibly to RA. Excess RA is inactivated by conversion to hydroxylated derivatives. Much is left to learn, especially about retinoid binding proteins and the trafficking of the hydrophobic retinoid substrates between membrane bound and cytosolic enzymes. Here, background on development of the field and an update on recent advances in our understanding of the enzymatic pathways and mechanisms that control the rate of RA production and degradation are presented with a focus on the many questions that remain unanswered.

Cyp26 Enzymes Facilitate Second Heart Field Progenitor Addition and Maintenance of Ventricular Integrity.

Although retinoic acid (RA) teratogenicity has been investigated for decades, the mechanisms underlying RA-induced outflow tract (OFT) malformations are not understood. Here, we show zebrafish embryos deficient for Cyp26a1 and Cyp26c1 enzymes, which promote RA degradation, have OFT defects resulting from two mechanisms: first, a failure of second heart field (SHF) progenitors to join the OFT, instead contributing to the pharyngeal arch arteries (PAAs), and second, a loss of first heart field (FHF) ventricular cardiomyocytes due to disrupted cell polarity and extrusion from the heart tube. Molecularly, excess RA signaling negatively regulates fibroblast growth factor 8a (fgf8a) expression and positively regulates matrix metalloproteinase 9 (mmp9) expression. Although restoring Fibroblast growth factor (FGF) signaling can partially rescue SHF addition in Cyp26 deficient embryos, attenuating matrix metalloproteinase (MMP) function can rescue both ventricular SHF addition and FHF integrity. These novel findings indicate a primary effect of RA-induced OFT defects is disruption of the extracellular environment, which compromises both SHF recruitment and FHF ventricular integrity.

Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency.

Mutations in the homeobox gene SHOX cause SHOX deficiency, a condition with clinical manifestations ranging from short stature without dysmorphic signs to severe mesomelic skeletal dysplasia. In rare cases, individuals with SHOX deficiency are asymptomatic. To elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with SHOX deficiency. The variant p.Phe508Cys of the retinoic acid catabolizing enzyme CYP26C1 co-segregated with the SHOX variant p.Val161Ala in the affected individuals, while the SHOX mutant alone was present in asymptomatic individuals. Two further cases with SHOX deficiency and damaging CYP26C1 variants were identified in a cohort of 68 individuals with LWD The identified CYP26C1 variants affected its catabolic activity, leading to an increased level of retinoic acid. High levels of retinoic acid significantly decrease SHOX expression in human primary chondrocytes and zebrafish embryos. Individual morpholino knockdown of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. Together, our findings describe CYP26C1 as the first genetic modifier for SHOX deficiency.

Hedgehog and retinoid signaling alters multiple myeloma microenvironment and generates bortezomib resistance.

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in bortezomib (BTZ) resistance. However, the mechanisms involved in these interactions are not completely understood. We previously showed that expression of CYP26 in BM stromal cells maintains a retinoic acid-low (RA-low) microenvironment that prevents the differentiation of normal and malignant hematopoietic cells. Since a low secretory B cell phenotype is associated with BTZ resistance in MM and retinoid signaling promotes plasma cell differentiation and Ig production, we investigated whether stromal expression of the cytochrome P450 monooxygenase CYP26 modulates BTZ sensitivity in the BM niche. CYP26-mediated inactivation of RA within the BM microenvironment prevented plasma cell differentiation and promoted a B cell-like, BTZ-resistant phenotype in human MM cells that were cocultured on BM stroma. Moreover, paracrine Hedgehog secretion by MM cells upregulated stromal CYP26 and further reinforced a protective microenvironment. These results suggest that crosstalk between Hedgehog and retinoid signaling modulates BTZ sensitivity in the BM niche. Targeting these pathological interactions holds promise for eliminating minimal residual disease in MM.

Expression of retinoic acid-metabolizing enzymes, ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1 in canine testis during post-natal development.

Mammalian spermatogenesis involves highly regulated temporal and spatial dynamics, carefully controlled by several signalling processes. Retinoic acid (RA) signalling could have a critical role in spermatogenesis by promoting spermatogonia differentiation, adhesion of germ cells to Sertoli cells, and release of mature spermatids. An optimal testicular RA concentration is maintained by retinaldehyde dehydrogenases (ALDHs), which oxidize RA precursors to produce RA, whereas the CYP26 class of enzymes catabolizes (oxidize) RA into inactive metabolites. The objective was to elucidate gene expression of these RA-metabolizing enzymes (ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1) and their protein presence in testes of young, peripubertal and adult dogs. Genes encoding RA-synthesizing isozymes ALDH1A1, ALDH1A2 and ALDH1A3 and RA-catabolizing isomers CYP26A1, CYP26B1 and CYP26C1 were expressed in testis at varying levels during testicular development from birth to adulthood in dogs. Based on detailed analyses of mRNA expression patterns, ALDH1A2 was regarded as a primary RA-synthesizing enzyme and CYP26B1 as a critical RA-hydrolysing enzyme; presumably, these genes have vital roles in maintaining RA homeostasis, which is imperative to spermatogenesis and other testicular functions in post-natal canine testis.

Elevated expression of the retinoic acid-metabolizing enzyme CYP26C1 in primary breast carcinomas.

Retinoic acid (RA)-metabolizing enzyme CYP26A1 has been shown to have increased expression levels in breast cancers and to effectively promote the survival of breast carcinoma cells, implying a potential oncogenic function. However, the expression of CYP26C1, another CYP26 family member, in primary breast carcinoma remains to be clarified. In the present study, we examined the expression of CYP26C1 by immunohistochemistry, using three different types of microarray, and observed strong cytoplasmic staining of CYP26C1 in 73 of the 219 (33.3 %) breast carcinomas. In contrast, CYP26C1 was not expressed in normal ductal and lobular cells in non-neoplastic tissue. Interestingly, increased expression of CYP26C1 was significantly associated with a high Ki-67 labeling index and a grade of tumor. However, CYP26C1 immunoreactivity was not associated with clinicopathological variables, including primary tumor status, lymph node involvement, distant metastasis, and tumor stage. In addition, CYP26C1 positivity was independent of the expression status of the hormone receptors and immunohistochemical surrogates for the intrinsic subtypes of breast cancer. This report is the first to demonstrate elevated expression of CYP26C1 in primary breast carcinomas. Based on the RA-catabolizing activity of CYP26C1, our data suggest that CYP26C1 expression may contribute to neoplasia in the breast.