CYP26A1 Links WNT and Retinoic Acid Signaling: A Target to Differentiate ALDH+ Stem Cells in APC-Mutant CRC.

APC mutation is the main driving mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. Hypotheses: A functional link exists between the WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induce wt-APC expression in APC-mutant CRC cells, and we assess the ability of all-trans retinoic acid (ATRA) to induce differentiation. We found that ATRA increased expression of the WNT target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, the RA and WNT pathways crosstalk to modulate CYP26A1, which metabolizes retinoids. Moreover, inducing wt-APC augments ATRA-induced cell differentiation by: (i) decreasing cell proliferation; (ii) suppressing ALDH1A1 expression; (iii) decreasing ALDH+ SCs; and (iv) increasing neuroendocrine cell differentiation. A novel CYP26A1-based network that links WNT and RA signaling was also identified by NanoString profiling/bioinformatics analysis. Furthermore, CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Notably, in wt-APC-CRCs, decreased CYP26A1 improved patient survival. These findings have strong potential for clinical translation.

Cytochrome P450 26A1 Contributes to the Maintenance of Neuropathic Pain.

The cytochrome P450 proteins (CYP450s) have been implicated in catalyzing numerous important biological reactions and contribute to a variety of diseases. CYP26A1, a member of the CYP450 family, carries out the oxidative metabolism of retinoic acid (RA), the active metabolite of vitamin A. Here we report that CYP26A1 was dramatically upregulated in the spinal cord after spinal nerve ligation (SNL). CYP26A1 was mainly expressed in spinal neurons and astrocytes. HPLC analysis displayed that the content of all-trans-RA (at-RA), the substrate of CYP26A1, was reduced in the spinal cord on day 7 after SNL. Inhibition of CYP26A1 by siRNA or inhibition of CYP26A1-mediated at-RA catabolism by talarozole relieved the SNL-induced mechanical allodynia during the maintenance phase of neuropathic pain. Talarozole also reduced SNL-induced glial activation and proinflammatory cytokine production but increased anti-inflammatory cytokine (IL-10) production. The RA receptors RARα, RXRß, and RXRγ were expressed in spinal neurons and glial cells. The promoter of Il-10 has several binding sites for RA receptors, and at-RA directly increased Il-10 mRNA expression in vitro. Finally, intrathecal IL-10 attenuated SNL-induced neuropathic pain and reduced the activation of astrocytes and microglia. Collectively, the inhibition of CYP26A1-mediated at-RA catabolism alleviates SNL-induced neuropathic pain by promoting the expression of IL-10 and suppressing glial activation. CYP26A1 may be a potential therapeutic target for the treatment of neuropathic pain.

Expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF in the developing rhesus monkey retina.

The fovea centralis (fovea) is a specialized region of the primate retina that plays crucial roles in high-resolution visual acuity and color perception. The fovea is characterized by a high density of cone photoreceptors and no rods, and unique anatomical properties that contribute to its remarkable visual capabilities. Early histological analyses identified some of the key events that contribute to foveal development, but the mechanisms that direct the specification of this area are not understood. Recently, the expression of the retinoic acid-metabolizing enzyme CYP26A1 has become a hallmark of some of the retinal specializations found in vertebrates, including the primate fovea and the high-acuity area in avian species. In chickens, the retinoic acid pathway regulates the expression of FGF8 to then direct the development of a rod-free area. Similarly, high levels of CYP26A1, CDKN1A, and NPVF expression have been observed in the primate macula using transcriptomic approaches. However, which retinal cells express these genes and their expression dynamics in the developing primate eye remain unknown. Here, we systematically characterize the expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF during the development of the rhesus monkey retina, from early stages of development in the first trimester until the third trimester (near term). Our data suggest that some of the markers previously proposed to be fovea-specific are not enriched in the progenitors of the rhesus monkey fovea. In contrast, CYP26A1 is expressed at high levels in the progenitors of the fovea, while it localizes in a subpopulation of macular Müller glia cells later in development. Together these data provide invaluable insights into the expression dynamics of several molecules in the nonhuman primate retina and highlight the developmental advancement of the foveal region.

CLMP is a tumor suppressor that determines all-trans retinoic acid response in colorectal cancer.

CAR-like membrane protein (CLMP) is a tight junction-associated protein whose mutation is associated with congenital short bowel syndrome (CSBS), but its functions in colorectal cancer (CRC) remain unknown. Here, we demonstrate that CLMP is rarely mutated but significantly decreased in CRC patients, and its deficiency accelerates CRC tumorigenesis, growth, and resistance to all-trans retinoic acid (ATRA). Mechanistically, CLMP recruits ß-catenin to cell membrane, independent of cadherin proteins. CLMP-mediated ß-catenin translocation inactivates Wnt(Wingless and INT-1)/ß-catenin signaling, thereby suppressing CRC tumorigenesis and growth in ApcMin/+, azoxymethane/dextran sodium sulfate (AOM/DSS), and orthotopic CRC mouse models. As a direct target of Wnt/ß-catenin, cytochrome P450 hydroxylase A1 (CYP26A1)-an enzyme that degrades ATRA to a less bioactive retinoid-is upregulated by CLMP deficiency, resulting in ATRA-resistant CRC that can be reversed by administering CYP26A1 inhibitor. Collectively, our data identify the anti-CRC role of CLMP and suggest that CYP26A1 inhibitor enable to boost ATRA's therapeutic efficiency.

Metformin reduces hepatocarcinogenesis by inducing downregulation of Cyp26a1 and CD8+ T cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer with major challenges in both prevention and therapy. Metformin, adenosine monophosphate-activated protein kinase (AMPK) activator, has been suggested to reduce the incidence of HCC when used for patients with diabetes in preclinical and clinical studies. However, the possible effects of metformin and their mechanisms of action in non-diabetic HCC have not been adequately investigated. METHODS: Fah-/-  mice were used to construct a liver-injury-induced non-diabetic HCC model for exploring hepatocarcinogenesis and therapeutic potential of metformin. Changes in relevant tumour and biochemical indicators were measured. Bulk and single-cell RNA-sequencing analyses were performed to validate the crucial role of proinflammatory/pro-tumour CD8+ T cells. In vitro and in vivo experiments were performed to confirm Cyp26a1-related antitumour mechanisms of metformin. RESULTS: RNA-sequencing analysis showed that chronic liver injury led to significant changes in AMPK-, glucose- and retinol metabolism-related pathways in Fah-/- mice. Metformin prevented the formation of non-diabetic HCC in Fah-/- mice with chronic liver injury. Cyp26a1 ddexpression in hepatocytes was significantly suppressed after metformin treatment. Moreover, downregulation of Cyp26a1 occurred in conjunction with increased levels of all-trans-retinoic acid (atRA), which is involved in the activation of metformin-suppressed hepatocarcinogenesis in Fah-/- mice. In contrast, both CD8+  T-cell infiltration and proinflammatory/pro-tumour cytokines in the liver were significantly upregulated in Fah-/- mice during chronic liver injury, which was notably reversed by either metformin or atRA treatment. Regarding mechanisms, metformin regulated the decrease in Cyp26a1 enzyme expression and increased atRA expression via the AMPK/STAT3/Gadd45ß/JNK/c-Jun pathway. CONCLUSIONS: Metformin inhibits non-diabetic HCC by upregulating atRA levels and downregulating CD8+ T cells. This is the first reporting that the traditional drug metformin regulates the metabolite atRA via the Cyp26a1-involved pathway. The present study provides a potential application of metformin and atRA in non-diabetic HCC.

Mechanism of thyroid hormone and its structurally similar contaminant bisphenol S exposure on retinoid metabolism in zebrafish larval eyes.

The photoreceptor necessitates the retinoids metabolism processes in visual cycle pathway to regenerate visual pigments and sustain vision. Bisphenol S (BPS), with similar structure of thyroid hormone (TH), was reported to impair the light-sensing function of zebrafish larvae via disturbing TH-thyroid hormone receptor ß (TRß) signaling pathway. However, it remains unknown whether TRß could modulate the toxicity of BPS on retinoid metabolism in visual cycle. This study showed that BPS diminished the optokinetic response of zebrafish larvae and had a stimulative effect on all-trans-retinoic acid (atRA) metabolism, like exogenous T3 exposure. By modulating CYP26A1 and TRß expression, it was found that CYP26A1 played a crucial role in catalyzing oxidative metabolism of atRA and retinoids regeneration in visual cycle, and TRß mediated cyp26a1 expression in zebrafish eyes. Similar with 10 nM T3 treatment, cyp26a1 expression could be induced by BPS in the presence of TRß. Further, in CYP26A1 and TRß- deficient eyes, 100 µg/L BPS could no longer promote atRA metabolism, or decrease the all-trans-retinol and 11-cis retinal contents in visual cycle, demonstrating that BPS exposure disturbed CYP26A1-mediated visual retinoids metabolism via TRß. Overall, this study highlights the role of TRß in mediating the retinoids homeostasis disruption caused by BPS, and provides new clues for exploring molecular targets of visual toxicity under pollutants stress.

Targeting the retinoic acid signaling pathway as a modern precision therapy against cancers.

Retinoic acid (RA) is a vital metabolite derived from vitamin A. RA plays a prominent role during development, which helps in embryological advancement and cellular differentiation. Mechanistically, RA binds to its definite nuclear receptors including the retinoic acid receptor and retinoid X receptor, thus triggering gene transcription and further consequences in gene regulation. This functional heterodimer activation later results in gene activation/inactivation. Several reports have been published related to the detailed embryonic and developmental role of retinoic acids and as an anti-cancer drug for specific cancers, including acute promyelocytic leukemia, breast cancer, and prostate cancer. Nonetheless, the other side of all-trans retinoic acid (ATRA) has not been explored widely yet. In this review, we focused on the role of the RA pathway and its downstream gene activation in relation to cancer progression. Furthermore, we explored the ways of targeting the retinoic acid pathway by focusing on the dual role of aldehyde dehydrogenase (ALDH) family enzymes. Combination strategies by combining RA targets with ALDH-specific targets make the tumor cells sensitive to the treatment and improve the progression-free survival of the patients. In addition to the genomic effects of ATRA, we also highlighted the role of ATRA in non-canonical mechanisms as an immune checkpoint inhibitor, thus targeting the immune oncological perspective of cancer treatments in the current era. The role of ATRA in activating independent mechanisms is also explained in this review. This review also highlights the current clinical trials of ATRA in combination with other chemotherapeutic drugs and explains the future directional insights related to ATRA usage.

Evidence for RA-dependent meiosis onset in a turtle embryo.

Meiotic entry is one of the earliest sex determination events of the germ cell in higher vertebrates. Although advances in meiosis onset have been achieved in mammals, birds and fish, how this process functions in reptiles is largely unknown. In this study, we present the molecular analysis of meiosis onset and the role of retinoic acid (RA) in this process in the red-eared slider turtle. Our results using Stra8 as a pre-meiosis indicator show that in the female embryonic gonad, meiosis commitment starts around stage 19. Additionally, signals of the meiosis marker Sycp3 could be detected at stage 19 and become highly expressed by stage 23. No expression of these genes was detected in male embryonic gonads, suggesting the entry into meiosis prophase I was restricted to female embryonic germ cells. Notably, RA activity in fetal gonads is likely to be elevated in females than that in males, as evidenced by the higher expression of RA synthase Aldh1a1 and lower expression of RA-degrading enzyme Cyp26a1 in female gonads prior to meiotic entry. In addition, exogenous RA treatment induced the expression of Stra8 and Sycp3 in both sexes, whether in vivo or in vitro. Together, these results indicate that high levels of RA in the embryonic female gonads can lead to the initiation of meiosis in the turtle.

Cyp26a1 supports postnatal retinoic acid homeostasis and glucoregulatory control.

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control.

Elevated expression of endocan in the development of cervical squamous neoplasia of the uterus.

Accumulated evidence has shown that endocan, which was originally called endothelial cell-specific molecule-1, is an attractive prognostic factor in a variety of cancers. However, the relevance of endocan expression in human malignancies remains to be clarified. In the present study, the expression of endocan in cervical squamous neoplasia of the uterus, including low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively), as well as in invasive squamous cell carcinoma was examined by immunohistochemistry. Endocan was not sufficiently expressed in the normal cervical epithelium. Endocan expression was present in LSIL cases but was limited to basal and parabasal areas of the cells. HSIL cases exhibited strong expression of endocan with widely distributed expression toward the epithelial surface. In contrast, further strong expression of endocan was not observed in patients with invasive carcinoma. This study is the first study showing increased expression of endocan in precancerous dysplastic lesions and malignancy of the cervix. The data suggest that a high expression level of endocan potentially contributes to the development of cervical squamous neoplasia of the uterus.

Retinoic Acid Signalling in the Pineal Gland Is Conserved across Mammalian Species and Its Transcriptional Activity Is Inhibited by Melatonin.

The pineal gland is integral to the circadian timing system due to its role in nightly melatonin production. Retinoic acid (RA) is a potent regulator of gene transcription and has previously been found to exhibit diurnal changes in synthesis and signalling in the rat pineal gland. This study investigated the potential for the interaction of these two systems. PCR was used to study gene expression in mouse and human pineal glands, ex-vivo organotypic cultured rat pineal gland and cell lines. The mouse and human pineal glands were both found to express the necessary components required for RA signalling. RA influences the circadian clock in the brain, therefore the short-term effect of RA on clock gene expression was determined in ex vivo rat pineal glands but was not found to rapidly regulate Per1, Per2, Bmal1, or Cry1. The interaction between RA and melatonin was also investigated and, unexpectedly, melatonin was found to suppress the induction of gene transcription by RA. This study demonstrates that pineal expression of the RA signalling system is conserved across mammalian species. There is no short-term regulation of the circadian clock but an inhibitory effect of melatonin on RA transcriptional activity was demonstrated, suggesting that there may be functional cross-talk between these systems.

In Vitro Transfection of Up-Regulated Genes Identified in Favorable-Outcome Neuroblastoma into Cell Lines.

We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence.

Development of an adenovirus-mediated reporter assay system to detect a low concentration of retinoic acid in MCF-7 cells.

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.

The multiciliated cells in Rathke's cleft express CYP26A1 and respond to retinoic acid in the pituitary.

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.

Cytochrome P450 26A1 regulates the clusters and killing activity of NK cells during the peri-implantation period.

Cytochrome P450 26A1 (CYP26A1) plays a vital role in early pregnancy in mice. Our previous studies have found that CYP26A1 affects embryo implantation by modulating natural killer (NK) cells, and that there is a novel population of CYP26A1+ NK cells in the uteri of pregnant mice. The aim of this study was to investigate the effects of CYP26A1 on the subsets and killing activity of NK cells. Through single-cell RNA sequencing (scRNA-seq), we identified four NK cell subsets in the uterus, namely, conventional NK (cNK), tissue-resident NK (trNK) 1 and 2, and proliferating trNK (trNKp). The two most variable subpopulations after uterine knockdown of CYP26A1 were trNKp and trNK2 cells. CYP26A1 knockdown significantly downregulated the expression of the NK cell function-related genes Cd44, Cd160, Vegfc, and Slamf6 in trNK2 cells, and Klra17 and Ogn in trNKp cells. Both RNA-seq and cytotoxicity assays confirmed that CYP26A1+ NK cells had low cytotoxicity. These results indicate that CYP26A1 may affect the immune microenvironment at the maternal-foetal interface by regulating the activity of NK cells.

Cytochrome P450 26A1 Modulates the Polarization of Uterine Macrophages During the Peri-Implantation Period.

Uterine M1/M2 macrophages activation states undergo dynamic changes throughout pregnancy, and inappropriate macrophages polarization can cause adverse pregnancy outcomes, especially during the peri-implantation period. Our previous studies have confirmed that Cytochrome P450 26A1 (CYP26A1) can affect embryo implantation by regulating uterine NK cells and DCs. The aim of this study was to investigate whether CYP26A1 regulates the polarization of uterine macrophages in early pregnancy. Here, we observed that Cyp26a1 was significantly upregulated in M1 as compared with M2 of uterine macrophages, Raw264.7 and iBMDM. Knockdown of CYP26A1 in mice uterine significantly decreased the number of embryo implantation sites and the proportion of CD45+F4/80+CD206 - M1-like uterine macrophages. Primary uterine macrophages treated with anti-CYP26A1 antibody expressed significantly lower levels of M1 markers Nos2, Il1b, Il6 and Tnf-a. In CYP26A1 knockout Raw264.7 cells, the protein levels of M1 markers TNF-α, IL-6 and CD86 were significantly decreased as compared with the wild type cells. Moreover, CYP26A1 deficiency decreased the ability to produce nitric oxide and increased the phagocytosis capacity of Raw264.7 cells under M1 stimulation state. The re-introduction of CYP26A1 partially reversed the polarization levels of M1 in CYP26A1 knockout Raw264.7 cells. CYP26A1 may regulate the polarization of uterine macrophages to M1 through Stap1 and Slc7a2. In summary, these results indicate that CYP26A1 plays a significant role in macrophage polarization, and knockdown of CYP26A1 can cause insufficient M1 polarization during the peri-implantation period, which has adverse effects on blastocyst implantation.

A novel Cytochrome P450 26A1 expressing NK cell subset at the mouse maternal-foetal interface.

Cyp26a1 had important roles in mouse embryo implantation and was highly expressed in some of NK cells at the human maternal-foetal interface in early pregnancy. However, the regulatory effect of Cyp26a1 on NK cells remains poorly understood. Through qPCR and flow cytometric assays, we found that Cyp26a1 was expressed by mouse uterine NK cells but not spleen NK cells during the peri-implantation period and there was a group of NK cells that highly expressed Cyp26a1, that is Cyp26a1+ NK cell subset. single cell-population transcriptome sequencing on Cyp26a1+ NK and Cyp26a1- NK cell subsets was performed. We found that there were 3957 differentially expressed genes in the Cyp26a1+ NK cell subset with a cut-off of fold change ≥2 and FDR < 0.01, 2509 genes were up-regulated and 1448 genes were down-regulated in Cyp26a1+ NK cell subset. Moreover, cytokine-cytokine receptor interaction signalling pathway and natural killer cell-mediated cytotoxicity signalling pathway were enriched according to KEGG pathway enrichment analysis. We further found that the expression of Gzma and Klrg1 was significantly increased and Fcgr4 was significantly decreased when inhibiting Cyp26a1. Our experimental results show that there is a novel NK cell subset of Cyp26a1+ NK cells in mouse uterus and Cyp26a1 can regulate the gene expression of Gzma, Klrg1 and Fcgr4 in the Cyp26a1+ NK cells.

Identification of a novel CYP26A1 mutation in a Chinese family with congenital microtia.

OBJECTIVES: Microtia is defined as a congenital malformation characterized by a small, abnormally shaped auricle, with atresia or stenosis of the auditory canal. This study investigated a mutation of the cytochrome P450, family 26, subfamily A, polypeptide 1(CYP26A1) gene, which is considered important in craniofacial development, in a family affected with microtia. METHODS: Whole-exome sequencing (WES) was performed on the proband and his family members to identify disease-associated variants. Computational predictions of the altered protein were analyzed using several bioinformatics tools. The wild-type (WT) and mutant forms of CYP26A1 cDNA were transfected into human embryonic kidney cells, and the mRNA and protein levels were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. RESULTS: In this two-generation family, the proband and his mother were diagnosed with unilateral microtia. Unilateral microtia and ipsilateral accessory ear were observed in one of the twins, who were sisters of the proband. The father and the other twin showed no abnormal clinical features. A heterozygous mutation of a C to T in the CYP26A1 gene, which leads to truncation of the CYP26A1 protein, was identified in this family. The nonsense mutation cosegregated with patients and was absent in normal members of the family. The prediction software indicated that it was a possibly pathogenic mutation. The structure of the protein varied significantly between the WT and mutant proteins. Functional analysis showed that this mutation caused a significant decrease in both the mRNA and protein levels. CONCLUSIONS: Our findings suggest that this mutation of CYP26A1 may be a pathogenic factor leading to the phenotypes of microtia and accessory ear in this family. Further studies are needed to prove the function of this mutation and to explore the possible mechanism by which this variant is involved in the occurrence of microtia.

CYP26A1 Is a Novel Biomarker for Betel Quid-Related Oral and Pharyngeal Cancers.

Betel quid (BQ) has been classified as a Group I human carcinogen in light of evidence demonstrating an association with an elevated risk of oral and pharyngeal cancers. To date, the incidence rate of oral and pharynx cancers among Taiwanese men ranks the highest worldwide. However, no study has yet confirmed variants of CYP26A1 was associated with the risks of oral and pharyngeal cancers. A case-control study was conducted (n = 339). CYP26A1 polymorphism was performed using SNP assay. Real-time qRT-PCR and Western blotting were used to determine the levels of CYP26A1 expression. The cancer cell model involved treatment with arecoline. Our findings showed that the downregulation of CYP26A1 mRNA and protein expression are more frequently observed in cancerous tissues than adjacent normal tissues in patients with oral and pharynx cancers (p < 0.01). We found that CYP26A1 was downregulated as the arecoline dose increased. We hypothesized that lower levels of CYP26A1 mRNA expression can be utilized a clinically biomarker causes oral and pharynx cancers. Arecoline appears to modulate CYP26A1 expression through specific pathways. Carriers of CYP26A1 SNP, rs2068888 (G/G)/rs4418728 (G/G) and who have lower levels of CYP26A1 expression are associated with an increased risk of oral and pharyngeal cancers.

Using the human CYP26A1 gene promoter as a suitable tool for the determination of RAR-mediated retinoid activity.

We have used a shortened construct form of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase (known as E4) or a red fluorescent protein, RFP (known as E4.2) as the reporter gene and examined their responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of at-RA in cells cotransfected with retinoic acid receptors (RAR). The promoter also responded quantitatively to retinol and various other retinoids. An isolated clonal line of HEK293T cells that was permanently transfected with the promoter driving the expression of RFP responded to both at-RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was also used to assess the retinoid activity of 3 novel synthetic retinoid analogues. Among them, EC23 was shown to be more potent than at-RA at lower concentrations and also more stable than at-RA. The promoter was also used to estimate the retinoid activities of intact rat serum samples as well as extracts of rat liver and lung, using retinol and at-RA as the reference standards. The retinoid activities could be measured in control rat serum samples and were increased in the serum of at-RA-treated rats. The total retinol and at-RA levels in the rat liver and lung samples determined by this promoter-based assay were compared with total retinol levels determined by the UPLC as the conventional methods. This system should offer a biologically-based alternative to mass-based retinoid analysis.