Cyp26b1 Expression Research Articles

FRI373 Effect Of Neonatal R115866 Injection On Ovary Development In Mice

Abstract Disclosure: M. Akroush: None. A. Urbanowski: None. A. Edwards: None. J. Kipp: None. The active metabolite of vitamin A, retinoic acid, is vital to several physiological functions and is most notably associated with vision and organ development. Its role in the male reproductive system is well understood, but studies on female reproduction are lacking. Recent studies show that Cyp26b1, a retinoic acid degrading enzyme, is expressed highly in the day 1-10 mouse ovary. This time point correlates with the establishment of the primordial follicle pool. In vitro studies show that both retinoic acid and R115866, a Cyp26b1 inhibitor that blocks retinoic acid metabolism, increase cell proliferation. Therefore, we hypothesize that retinoic acid plays a critical role in the early development of the ovary. To test this hypothesis, we injected neonatal mice with R115866 at either 0.5 or 1.0 mg/kg during the first 7 days after birth when the levels of Cyp26b1 expression are high. At day 19, week 7, and week 15, ovaries were harvested for morphological, immunohistochemical, and RT-PCR studies. Our results showed that animals injected with R115866 showed dose-dependent decreases in body weight in day 19 and week 7 mice, while week 15 mice appeared to recover. Neonatal R115866 injection increased the number of multi-nucleic oocytes at day 19 and bursa cysts at week 15. Cyp26b1 mRNA and protein levels were both increased by R115866 treatment in Day 19 ovaries, as confirmed by real-time RT PCR and immunohistochemical studies, consistent with the effect of these inhibitors in increasing RA levels which in turn induce Cyp26b1 expression. Neonatal R115866 injection also increased the expression of proliferative markers, including Ki67, Cyclin E1, and PCNA. Currently, we are completing studies on follicle classification and count. Overall, our results suggest that retinoic acid plays an important role in the development of the mouse ovary and that balanced retinoic acid levels in early development are needed for the health of ovaries. Presentation: Friday, June 16, 2023

Identification and Expression Pattern of cyp26b1 Gene in Gonad of the Chinese Tongue Sole (Cynoglossus semilaevis).

Simple SummaryIn fish, it is obvious that the asynchronous development of the gonads and sexual dimorphism limit the development of aquaculture, so the research into sex-differentiation and gonadal growth is very important. Due to the sexual reversal phenomenon (genetic females becoming phenotypic males), the Chinese tongue sole (Cynoglossus semilaevis) is a great model for investigating sex-differentiation. Herein, we report one gene involved in sex-differentiation and gonadal growth of the Chinese Tongue Sole. The gene cyp26b1 (cytochrome P450 family 26 subfamily b member 1) is a metabolizing Retinoic Acid (RA) enzyme. Since it regulates RA to control sex determination and differentiation, cyp26b1 is considered a critical part of mammals’ ovary-antagonizing and testis-determining downstream passageway of Sry (sex-determining region Y) and Sox9 (sry- box transcription factor 9). In fish, the related research is reported only on the Japanese flounder (Paralichthys olivaceus) and zebrafish (Danio rerio). In the current investigation, the identification and expression pattern of the cyp26b1 gene in the Chinese tongue sole suggested that cyp26b1 might impact sex-differentiation and gonadal development.As an RA-metabolizing enzyme, cyp26b1 has a substantial impact on RA-signaling pathways. The cyp26b1 gene from the Chinese tongue sole was cloned and identified in this investigation. The cyp26b1 ORF was 1536 bp in length and encoded a 512 amino acid protein. A quantitative real-time PCR (qPCR) indicated that the cyp26b1 expression is no significant sexual dimorphism in the gonads at the 80 days post-hatching (dph) stages. After 4 months post-hatching (mph), the expression of cyp26b1 showed sexual dimorphism and lower level of expression in the ovaries than in the testes. An in situ hybridization demonstrated that cyp26b1 mRNA was primarily located in the testis. Interestingly, the cyp26b1 mRNA probe was also detected in the ovaries. These results suggested that cyp26b1 participates in the sex-differentiation and gonadal development of the Chinese tongue sole.

Retinoic acid levels control tooth morphology in fish

Misshapen teeth are highly common in humans. In most instances, they are due to genetic mutations in genes controlling the morphogenesis of teeth. This is the case for the Runx2 gene, which once mutated leads to cleidocranial dysplasia (CCD) an autosomal dominant genetic syndrome presenting with peg‐like teeth in humans. While many factors have been identified in the process of tooth morphogenesis, currently, little is known about how cell signaling participates in establishing organ shape during odontogenesis. Our previous work has identified the signaling molecule retinoic acid (RA) to be one of the main actors of tooth induction in fish and recent data suggest that RA also plays a role during tooth morphogenesis. Fish and zebrafish, in particular, are good models to study the genetics, cell and molecular biology, and organogenesis of the tooth in vertebrates. The objective of the proposed studies is to understand the roles played by retinoic acid during tooth morphogenesis in different fish species, to develop a fish model of CCD, to clarify the role played by Runx2 during tooth morphogenesis and to understand its link with RA signaling. By exposing fish embryos to exogenous RA and to an RA inhibitor during tooth morphogenesis we have been able to quantify changes in tooth shape and better understand the mechanism of action of RA signaling during tooth morphogenesis in fish. Exogenous RA exposure alters the shape of the first developing tooth in zebrafish making it narrower and more elongated resembling other tooth shape observed in the wild. RA exposure to a fish species, like the goldfish, with shorter and wider tooth makes it also narrower and more elongated making it more like a zebrafish tooth. At the molecular levels, the expression of tooth epithelium and mesenchyme markers dlx2a; dlx3b; pitx2a; runx2b and lhx6 is expanded in RA treated zebrafish embryo. Our data reveal that the levels of RA in different cells of the tooth germ is controlled by the timing and level of expression of the RA degrading enzyme cyp26b1 in a subset of cells of the developing tooth germ. We hypothesize that modifying the onset of cyp26b1 expression in the tooth germ will, therefore, change the level of RA available in the tooth germ, ultimately altering the shape of the tooth. Finally, our work has revealed more about how RA regulates Runx2 and controls tooth morphogenesis in the development of CCD and during evolution. Our project has revealed that fine tuning of RA level in the developing tooth is a crucial player in the vast tooth diversity observed in fish species in the wild.

Isoflavones Suppress Cyp26b1 Expression in the Murine Colonic Lamina Propria.

Isoflavones have many biological activities and are major bioactive components of kakkonto, a traditional Japanese herbal medicine. We previously reported that the combined therapy of oral immune therapy (OIT) and kakkonto downregulates the mRNA expression of Cyp26b1, a major retinoic acid (RA)-degrading enzyme, in the colon of food allergy mice and thereby ameliorates allergic symptoms. In this study, we evaluated the effects of various isoflavones on Cyp26b1 expression in primary cultured lamina propria (LP) cells isolated from the mouse colon. The mRNA expression of Cyp26b1 was extremely downregulated by all isoflavones tested in the LP cells except for puerarin. In particular, genistein and genistin markedly suppressed Cyp26b1 mRNA expression without affecting RA-synthesizing enzyme expression. Moreover, to evaluate the effects of isoflavones on allergic reactions, genistein and genistin were administered to ovalbumin (OVA)-induced food allergy mice. Oral administration of genistin suppressed the development of allergic symptoms. These results raise the possibility that isoflavones elevated the level of RA in the colon by inhibiting RA degradation and then the high concentration of RA in the colon might exert immunosuppressive and antiallergic effects on food allergy mice.

Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation

Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation

Retinoic acid synthesis and autoregulation mediate zonal patterning of vestibular organs and inner ear morphogenesis.

Retinoic acid (RA), a vitamin A (retinol) derivative, has pleiotropic functions during embryonic development. The synthesis of RA requires two enzymatic reactions: oxidation of retinol into retinaldehyde by alcohol dehydrogenases (ADHs) or retinol dehydrogenases (RDHs); and oxidation of retinaldehyde into RA by aldehyde dehydrogenases family 1, subfamily A (ALDH1as), such as ALDH1a1, ALDH1a2 and ALDH1a3. Levels of RA in tissues are regulated by spatiotemporal expression patterns of genes encoding RA-synthesizing and -degrading enzymes, such as cytochromeP450 26 (Cyp26 genes). Here, we show that RDH10 is important for both sensory and non-sensory formation of the vestibule of the inner ear. Mice deficient in Rdh10 exhibit failure of utricle-saccule separation, otoconial formation and zonal patterning of vestibular sensory organs. These phenotypes are similar to those of Aldh1a3 knockouts, and the sensory phenotype is complementary to that of Cyp26b1 knockouts. Together, these results demonstrate that RDH10 and ALDH1a3 are the key RA-synthesis enzymes involved in vestibular development. Furthermore, we discovered that RA induces Cyp26b1 expression in the developing vestibular sensory organs, which generates the differential RA signaling required for zonal patterning.

Increased Retinoic Acid Catabolism in Olfactory Sensory Neurons Activates Dormant Tissue-Specific Stem Cells and Accelerates Age-Related Metaplasia.

The cellular and molecular basis of metaplasia and declining neurogenesis in the aging olfactory epithelium (OE) remains unknown. The horizontal basal cell (HBC) is a dormant tissue-specific stem cell presumed to only be forced into self-renewal and differentiation by injury. Here we analyze male and female mice and show that HBCs also are activated with increasing age as well as non-cell-autonomously by increased expression of the retinoic acid-degrading enzyme CYP26B1. Activating stimuli induce HBCs throughout OE to acquire a rounded morphology and express IP3R3, which is an inositol-1,4,5-trisphosphate receptor constitutively expressed in stem cells of the adjacent respiratory epithelium. Odor/air stimulates CYP26B1 expression in olfactory sensory neurons mainly located in the dorsomedial OE, which is spatially inverse to ventrolateral constitutive expression of the retinoic acid-synthesizing enzyme (RALDH1) in supporting cells. In ventrolateral OE, HBCs express low p63 levels and preferentially differentiate instead of self-renewing when activated. When activated by chronic CYP26B1 expression, repeated injury, or old age, ventrolateral HBCs diminish in number and generate a novel type of metaplastic respiratory cell that is RALDH- and secretes a mucin-like mucus barrier protein (FcγBP). Conversely, in the dorsomedial OE, CYP26B1 inhibits injury-induced and age-related replacement of RALDH- supporting cells with RALDH1+ ciliated respiratory cells. Collectively, these results support the concept that inositol-1,4,5-trisphosphate type 3 receptor signaling in HBCs, together with altered retinoic acid metabolism within the niche, promote HBC lineage commitment toward two types of respiratory cells that will maintain epithelial barrier function once the capacity to regenerate OE cells ceases.SIGNIFICANCE STATEMENT Little is known about signals that activate dormant stem cells to self-renew and regenerate odor-detecting neurons and other olfactory cell types after loss due to injury, infection, or toxin exposure in the nose. It is also unknown why the stem cells do not prevent age-dependent decline of odor-detecting neurons. We show that (1) stem cells are kept inactive by the vitamin A derivative retinoic acid, which is synthesized and degraded locally by olfactory cells; (2) old age as well as repeated injuries activate the stem cells and exhaust their potential to produce olfactory cells; and (3) exhausted stem cells alter the local retinoic acid metabolism and maintain the epithelial tissue barrier by generating airway cells instead of olfactory cells.

CYP26B1 declines postnatally in Sertoli cells independently of androgen action in the mouse testis.

Meiosis begins at puberty and relies on several factors, including androgens and retinoic acid in the mouse testis. CYP26B1 degrades retinoic acid in the testis during prenatal development preventing meiosis initiation. Given the concurrence of meiotic entry and completion of Sertoli cell maturation in response to androgens at puberty in the mouse, we proposed that CYP26B1 is downregulated by androgens in the Sertoli cell during this period. By immunohistochemistry, we showed that CYP26B1 declines in Sertoli cells after birth. However, luciferase reporter assays and quantitative reverse transcription-polymerase chain reaction performed in the prepubertal mouse Sertoli cell line SMAT1 revealed no changes in Cyp26b1 expression in response to androgen treatment. Furthermore, studies carried out using primary Sertoli cells of 10-day-old mice showed no changes in either Cyp26b1 or CYP26B1 expression in response to androgen treatment. In summary, the hereby reported decline in CYP26B1 expression in Sertoli cells towards pubertal onset does not appear to be caused by a direct inhibitory effect of androgens on Sertoli cells in the mouse.

Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) regulates the concentration of all-trans retinoic acid (RA) and plays a key role in germ cell differentiation by controlling local distribution of RA. The mechanisms regulating Cyp26b1 expression in postnatal Sertoli cells, the main components of the stem cell niche, are so far unknown. During gonad development, expression of Cyp26b1 is maintained by Steroidogenic Factor 1 (SF-1) and Sex-Determining Region Y Box-9 (SOX9), which ensure that RA is degraded and germ cell differentiation is blocked. Here, we show that the NOTCH target Hairy/Enhancer-of-Split Related with YRPW Motif 1 (HEY1), a transcriptional repressor, regulates germ cell differentiation via direct binding to the Cyp26b1 promoter and thus inhibits its expression in Sertoli cells. Further, using in vivo germ cell ablation, we demonstrate that undifferentiated type A spermatogonia are the cells that activate NOTCH signaling in Sertoli cells through their expression of the NOTCH ligand JAGGED-1 (JAG1) at stage VIII of the seminiferous epithelium cycle, therefore mediating germ cell differentiation by a ligand concentration-dependent process. These data therefore provide more insights into the mechanisms of germ cell differentiation after birth and potentially explain the spatiotemporal RA pulses driving the transition between undifferentiated to differentiating spermatogonia.-Parekh, P. A., Garcia, T. X., Waheeb, R., Jain, V., Gandhi, P., Meistrich, M. L., Shetty, G., Hofmann, M.-C. Undifferentiated spermatogonia regulate Cyp26b1 expression through NOTCH signaling and drive germ cell differentiation.

Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression

Angulated femurs are present prenatally both in CYP26B1 deficient humans with a reduced capacity to degrade retinoic acid (RA, the active metabolite of vitamin A), and mice overexpressing vascular endothelial growth factor a (Vegfa). Since excessive ingestion of vitamin A is known to induce spontaneous fractures and as the Vegfa-induced femur angulation in mice appears to be caused by intrauterine fractures, we analyzed bones from a CYP26B1 deficient human and rats with hypervitaminosis A to further explore Vegfa as a mechanistic link for the effect of vitamin A on bone. We show that bone from a human with CYP26B1 mutations displayed periosteal osteoclasts in piles within deep resorption pits, a pathognomonic sign of hypervitaminosis A. Analysis of the human angulated fetal femur revealed excessive bone formation in the marrow cavity and abundant blood vessels. Normal human endothelial cells showed disturbed cell-cell junctions and increased CYP26B1 and VEGFA expression upon RA exposure. Studies in rats showed increased plasma and tissue Vegfa concentrations and signs of bone marrow microhemorrhage on the first day of excess dietary vitamin A intake. Subsequently hypervitaminosis A rats displayed excess bone formation, fibrosis and an increased number of megakaryocytes in the bone marrow, which are known characteristics of Vegfa overexpression. This study supports the notion that the skeletal phenotype in CYP26B1 deficient human bone is caused by excess RA. Our findings suggest that an initial part of the vitamin A mechanism causing bone alterations is mediated by excess Vegfa and disturbed bone marrow microvessel integrity.

Loss of Angelman Syndrome Protein E6AP Disrupts a Novel Antagonistic Estrogen-Retinoic Acid Transcriptional Crosstalk in Neurons.

Angelman syndrome (AS) is a complex genetic disorder that affects the nervous system. AS affects an estimated 1 in 12,000 to 20,000 individuals. Characteristic features of AS includes developmental delay or intellectual disability, severe speech impairment, seizures, small head size (microcephaly), and problems with movement and balance (ataxia). AS individuals usually have microdeletion of the maternal copy of 15q11.2-15q13 region of chromosome 15. The E6-associated protein (E6AP, an E3 ubiquitin protein ligase enzyme) is encoded by the gene UBE3A, which is located in this region, and it has been shown that deregulation of E6AP gives rise to AS and neuropathology of autism spectrum disorders (ASDs) (e.g., autism and Rett syndromes). We have shown that E6AP also acts as a coactivator of the estrogen receptor (ER). ER is a ligand-induced transcription factor that exerts potent and wide-ranging effects on the developing brain. Furthermore, the expression pattern of ER in the brain overlaps with that of E6AP. Up till now, all the published studies have examined the role of the ubiquitin-protein ligase activity of E6AP in the development of AS, and it is not known what role the newly discovered coactivation functions of E6AP and ER plays in the pathology of AS. Here, we demonstrate that E6AP and ER co-immunoprecipitate and are in the same protein complex in neuronal cells (Neuro2a). In addition, both colocalize in nuclear and cytoplasmic compartments of the mouse hippocampal neurons and Neuro2a cells. Moreover, we identified a novel E6AP and ER direct transcriptional regulation of a gene Cyp26b1 known to be involved in learning and memory processes. This transcriptional regulation involves recruitment of E6AP and ER to a newly discovered functional estrogen response element (ERE) located at the Cyp26b1 gene promoter and is associated with transcription permissive epigenetic events leading to increase of active transcription of the gene in neurons upon estrogen treatment. This novel transcriptional regulation was also validated in the AS mouse model where E6AP expression is abrogated in the mouse brain. In fact, Cyp26b1 expression is decreased by 31% in AS mice versus age-matched control (Ctrl) mice hippocampi. Also, retinoic acid transcriptional signaling was shown to be amplified as evidenced by specific increased Rarβ and decreased Erbb4 mRNA expression in AS mice versus Ctrl mice hippocampi. These transcript level changes were also supported by the same trend of changes at the protein level. Collectively, our data present a proof of principle that the transcriptional coactivation function of E6AP may have a crucial role in the pathobiology of AS. This function, yet to be thoroughly investigated, reveals the possibility of harnessing the antagonistic estrogen-retinoic acid transcriptional signaling crosstalk and potentially other unknown effectors for the investigation of important possible targets as putative novel treatment modalities and venues for reversing neurological manifestations in AS and related syndromes like ASDs.

Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development.

Zebrafish mutants with increased retinoic acid (RA) signaling due to the loss of the RA-inactivating enzyme Cyp26b1 develop a hyper-mineralized spine with gradually fusing vertebral body precursors (centra). However, the underlying cellular mechanisms remain incompletely understood. Here, we show that cells of the notochord epithelium named chordoblasts are sensitive to RA signaling. Chordoblasts are uniformly distributed along the anteroposterior axis and initially generate the continuous collagenous notochord sheath. However, subsequently and iteratively, subsets of these cells undergo further RA-dependent differentiation steps, acquire a stellate-like shape, downregulate expression of the collagen gene col2a1a, switch on cyp26b1 expression and trigger metameric sheath mineralization. This mineralization fails to appear upon chordoblast-specific cell ablation or RA signal transduction blockade. Together, our data reveal that, despite their different developmental origins, the activities and regulation of chordoblasts are very similar to those of osteoblasts, including their RA-induced transition from osteoid-producing cells to osteoid-mineralizing ones. Furthermore, our data point to a requirement for locally controlled RA activity within the chordoblast layer in order to generate the segmented vertebral column.

In vivo zebrafish morphogenesis shows Cyp26b1 promotes tendon condensation and musculoskeletal patterning in the embryonic jaw.

Integrated development of diverse tissues gives rise to a functional, mobile vertebrate musculoskeletal system. However, the genetics and cellular interactions that drive the integration of muscle, tendon, and skeleton are poorly understood. In the vertebrate head, neural crest cells, from which cranial tendons derive, pattern developing muscles just as tendons have been shown to in limb and trunk tissue, yet the mechanisms of this patterning are unknown. From a forward genetic screen, we determined that cyp26b1 is critical for musculoskeletal integration in the ventral pharyngeal arches, particularly in the mandibulohyoid junction where first and second arch muscles interconnect. Using time-lapse confocal analyses, we detail musculoskeletal integration in wild-type and cyp26b1 mutant zebrafish. In wild-type fish, tenoblasts are present in apposition to elongating muscles and condense in discrete muscle attachment sites. In the absence of cyp26b1, tenoblasts are generated in normal numbers but fail to condense into nascent tendons within the ventral arches and, subsequently, muscles project into ectopic locales. These ectopic muscle fibers eventually associate with ectopic tendon marker expression. Genetic mosaic analysis demonstrates that neural crest cells require Cyp26b1 function for proper musculoskeletal development. Using an inhibitor, we find that Cyp26 function is required in a short time window that overlaps the dynamic window of tenoblast condensation. However, cyp26b1 expression is largely restricted to regions between tenoblast condensations during this time. Our results suggest that degradation of RA by this previously undescribed population of neural crest cells is critical to promote condensation of adjacent scxa-expressing tenoblasts and that these condensations are subsequently required for proper musculoskeletal integration.

Stage-Specific Expression of CYP26B1 in the Adult Testis is Responsible for Pulsatile Retinoic Acid Signaling in Spermatogenesis

Retinoic acid (RA) plays important roles in spermatogenesis process; however, mechanisms underlying pulsatile RA signaling in meiotic initiation has not been understood yet. We studied expression pattern of RA-degrading enzyme CYP26B1 during development of rhesus monkey testis using qPCR and IHC. In developing monkey testis, the CYP26B1 protein was detected in cytoplasm of undifferentiated spermatogonia. Expression level of CYP26B1 mRNA was down-regulated during testis development which is consistent with initiation of meiosis in adult testis. In adult testes, a heterogeneous pattern of CYP26B1 expression was observed along different stages of seminiferous epithelium, presumably indicates expression pattern of protein is stage specific. Highest level of CYP26B1 protein expression was coincided with onset of meiosis and observed in early meiotic spermatocytes within seminiferous epithelial stages X-XII. Whereas, lowest level of CYP26B1 expression was observed in stages VI-IX of seminiferous epithelium, where undifferentiated Type A spermatogonia divide and differentiate to Type B spermatogonia, meiosis initiates and spermiogenesis occurs. This observations led us to suggest CYP26B1 might be responsible for pulsatile RA signaling in spermatogenesis. Findings presumably support that elevated amount of RA in undifferentiated Type A spermatogonia during stages VI-IX of the seminiferous epithelium of adult monkey testis may be responsible for differentiation of spermatogonia and their meiotic entry

Expression of STRA8 is conserved in therian mammals but expression of CYP26B1 differs between marsupials and mice.

The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown.

Abstract 4924: Adh1blow/Cyp26b1high activity of CD90+ (myo)fibroblasts supports tumor-promoting inflammation in colorectal cancer

Abstract Introduction. Retinol (RO) and its active metabolite, all-trans retinoic acid (atRA), have been widely studied as cancer chemotherapeutic agents. atRA is a critical immunoregulatory molecule reported to decrease cancer cell proliferation and inhibit tumor-promoting inflammation. However, recent studies report a loss of efficacy of atRA treatment to counteract colorectal cancer (CRC) progress, and the mechanisms underlying this failure are not fully understood. Alcohol dehydrogenase 1B (ADH1B), a major isoform of alcohol dehydrogenase expressed in normal colon, is responsible for the conversion of RO to atRA. We recently reported that stromal (myo)fibroblasts are major ADH1B expressers in normal colonic mucosa and that this expression is lost during the neoplastic transformation process of CRC. Thus, we hypothesized that aberrant retinoid signaling in cancer-associated fibroblasts (CAFs) is among key processes supporting tumor-promoting inflammation in CRC. Methods. Real-time PCR, confocal microscopy and cytokine luminex arrays were used to evaluate alterations in retinoid pathway gene expression and activity to define its relevance to the tumor-associated inflammation in human normal and CRC colonic tissues, and in normal primary (myo)fibroblasts (N-CMFs) and CAFs. Results. We observed a strong downregulation of ADH1B expression in CAFs in situ and in culture along with an increased expression of the tumor growth promoting inflammatory cytokine IL-6. Addition of the ADH1B substrate, RO, or its metabolite, atRA, to N-CMF cultures decreased the LPS-inducible IL-6 expression by N-CMFs. No downregulation of LPS-inducible IL-6 expression by RO was observed in CAFs, while atRA treatment partially inhibits IL-6 expression. Silencing of adh1b gene in N-CMFs led to the increased production of the LPS-inducible IL-6. This suggests that lack of ADH1B expression in CAFs prevents conversion of RO into atRA, resulting in the disruption of the negative regulation of IL-6. A decrease in downstream enzyme ALDH1A expression, which converts ADH1B’s byproduct retinaldehyde, to atRA, was also observed in CAFs. Finally, only a partial inhibition of IL-6 production by exogenous atRA was observed in CAFs. When compared to N-CMFs, CAFs expressed higher levels of CYP26B1, a cytochrome that is responsible for atRA degradation. Treatment of CAFs with atRA resulted in an eight fold higher upregulation of CYP26B1 expression than in N-CMFs. Conclusions. Taken together, our data suggests that CYP26B1 overexpression in CAFs along with ADH1B downregulation support aberrant retinoic acid metabolism in CRC. Adh1blow/Cyp26b1high activity of CAFs leads to a decrease of atRA availability in the tumor microenvironment, supporting IL-6-driven tumor growth promoting inflammation and resistance to exogenous atRA as a chemotherapeutic agent in CRC. Citation Format: Romain Villeger, Gabriella Uribe, Judy A. Trieu, Paul Johnson, Suimmin Qiu, Don W. Powell, Ellen J. Beswick, Iryna V. Pinchuk. Adh1blow/Cyp26b1high activity of CD90+ (myo)fibroblasts supports tumor-promoting inflammation in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4924. doi:10.1158/1538-7445.AM2017-4924

Activity-dependent and graded BACE1 expression in the olfactory epithelium is mediated by the retinoic acid metabolizing enzyme CYP26B1.

It is well established that environmental influences play a key role in sculpting neuronal connectivity in the brain. One example is the olfactory sensory map of topographic axonal connectivity. While intrinsic odorant receptor signaling in olfactory sensory neurons (OSN) determines anterior-posterior counter gradients of the axonal guidance receptors Neuropilin-1 and Plexin-A1, little is known about stimulus-dependent gradients of protein expression, which correlates with the functional organization of the olfactory sensory map along its dorsomedial (DM)-ventrolateral (VL) axis. Deficiency of the Alzheimer's β-secretase BACE1, which is expressed in a DM(low)-VL(high) gradient, results in OSN axon targeting errors in a DM>VL and gene dose-dependent manner. We show that expression of BACE1 and the all-trans retinoic acid (RA)-degrading enzyme Cyp26B1 form DM-VL counter gradients in the olfactory epithelium. Analyses of mRNA and protein levels in OSNs after naris occlusion, in mice deficient in the olfactory cyclic nucleotide-gated channel and in relation to onset of respiration, show that BACE1 and Cyp26B1 expression in OSNs inversely depend on neuronal activity. Overexpression of a Cyp26B1 or presence of a dominant negative RA receptor transgene selectively in OSNs, inhibit BACE1 expression while leaving the DM(low)-VL(high) gradient of the axonal guidance protein Neuropilin-2 intact. We conclude that stimulus-dependent neuronal activity can control the expression of the RA catabolic enzyme Cyp26B1 and downstream genes such as BACE1. This result is pertinent to an understanding of the mechanisms by which a topographic pattern of connectivity is achieved and modified as a consequence of graded gene expression and sensory experience.

The Expression and Prognostic Significance of Retinoic Acid Metabolising Enzymes in Colorectal Cancer

Colorectal cancer is one of the most common types of cancer with over fifty percent of patients presenting at an advanced stage. Retinoic acid is a metabolite of vitamin A and is essential for normal cell growth and aberrant retinoic acid metabolism is implicated in tumourigenesis. This study has profiled the expression of retinoic acid metabolising enzymes using a well characterised colorectal cancer tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosal samples. Immunohistochemistry was performed on the tissue microarray using monoclonal antibodies which we have developed to the retinoic acid metabolising enzymes CYP26A1, CYP26B1, CYP26C1 and lecithin retinol acyl transferase (LRAT) using a semi-quantitative scoring scheme to assess expression. Moderate or strong expression of CYP26A1was observed in 32.5% of cancers compared to 10% of normal colonic epithelium samples (p<0.001). CYP26B1 was moderately or strongly expressed in 25.2% of tumours and was significantly less expressed in normal colonic epithelium (p<0.001). CYP26C1 was not expressed in any sample. LRAT also showed significantly increased expression in primary colorectal cancers compared with normal colonic epithelium (p<0.001). Strong CYP26B1 expression was significantly associated with poor prognosis (HR = 1.239, 95%CI = 1.104–1.390, χ2 = 15.063, p = 0.002). Strong LRAT was also associated with poorer outcome (HR = 1.321, 95%CI = 1.034–1.688, χ2 = 5.039, p = 0.025). In mismatch repair proficient tumours strong CYP26B1 (HR = 1.330, 95%CI = 1.173–1.509, χ2 = 21.493, p<0.001) and strong LRAT (HR = 1.464, 95%CI = 1.110–1.930, χ2 = 7.425, p = 0.006) were also associated with poorer prognosis. This study has shown that the retinoic acid metabolising enzymes CYP26A1, CYP26B1 and LRAT are significantly overexpressed in colorectal cancer and that CYP26B1 and LRAT are significantly associated with prognosis both in the total cohort and in those tumours which are mismatch repair proficient. CYP26B1 was independently prognostic in a multivariate model both in the whole patient cohort (HR = 1.177, 95%CI = 1.020–1.216, p = 0.026) and in mismatch repair proficient tumours (HR = 1.255, 95%CI = 1.073–1.467, p = 0.004).

Characterization of zebrafish mutants with defects in bone calcification during development

Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line, bone calcification slow (bcs), which showed delayed axial vertebra calcification during development. Before 4–5days post-fertilization (dpf), the bcs embryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5–6dpf, when vertebrae calcification starts, bcs embryos began to show defects. At 7dpf, for example, in most of the bcs embryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2–14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed in bcs mutants when examined at 9 and 11dpf, respectively. Interestingly, by 13dpf the defects in bcs mutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g., Sox9b, Bmp2b, and Cyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases in Sox9b expression, no alterations in Bmp2b expression, but significant increases in Cyp26b1 expression. Together, the data suggest that bcs delays axial skeletal calcification, but does not affect bone formation and maturation.

The Retinoic Acid-Metabolizing Enzyme Cyp26b1 Regulates CD4 T Cell Differentiation and Function

The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4+ T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 −/− mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iTreg) cell- and TH17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iTreg and TH17 cells. Transfer of naïve Cyp26b1 −/− CD4+ T cells into Rag1 −/− mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.