Cloning of rat cytochrome P450RAI (CYP26) cDNA and regulation of its gene expression by all-trans-retinoic acid in vivo

A novel retinoic acid (RA)-inducible cytochrome P450 (P450 RAI or CYP26), previously cloned from human, zebra fish, and mouse, functions in the metabolism of all-trans-RA to polar metabolites including 4-hydroxy-RA and 4-oxo-RA. To further study CYP26 in the rat model, we first cloned rat CYP26 cDNA. The nucleotide sequence predicts a 497-amino-acid protein whose sequence is 95% identical to mouse and 91% homologous to human CYP26. Animal studies showed that CYP26 mRNA expression is very low (0.01+/-0.008;P<0.05) in vitamin-A-deficient rats compared to pair-fed vitamin-A-sufficient rats (defined as 1.0). In a kinetic study, vitamin-A-deficient rats were treated with approximately 100 microg of all-trans-RA and liver was collected after 3-72 h for analysis of CYP26 mRNA by quantitative real-time PCR. Liver CYP26 mRNA increased to nearly 10-fold above control after 3 h (P<0.01), reaching a peak of about 2000-fold greater around 10 h (P<0.001) and then decreased rapidly. The CYP26 dose response to RA was nearly linear (R(2)=0.9638). Additionally, significant regulation of CYP26 gene expression was observed in the vitamin-A-deficient, control, and RA-treated condition in lung, testis, and small intestine. We conclude that CYP26 mRNA expression is dynamically regulated in vivo by diet and RA in hepatic and extrahepatic tissues. The long-term down-regulation of CYP26 in retinoid deficiency may be critical for conserving RA, while the acute up-regulation of CYP26 may be important for preventing a deleterious overshoot of RA derived from either dietary or exogenous sources.     

Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid

Lecithin:retinol acyltransferase (LRAT), present in microsomes, catalyzes the transfer of the sn-1 fatty acid of phosphatidylcholine to retinol bound to a cellular retinol-binding protein. In the present study we have cloned mouse and rat liver LRAT cDNA and tested the hypothesis that LRAT mRNA, like LRAT activity, is regulated physiologically in a liver-specific manner. The nucleotide sequences of mouse and rat liver LRAT cDNA each encode a 231-amino acid protein with 94% similarity between these species, and approximately 80% similarity to a cDNA for LRAT from human retinal pigment epithelium. Expression of rat LRAT cDNA in HEK293T cells resulted in functional retinol esterification and storage. RNA from several rat tissues hybridized with liver LRAT cDNA. However, LRAT mRNA was virtually absent from the liver of vitamin A-deficient animals, while being unaffected in intestine and testis. LRAT mRNA was rapidly induced by retinoic acid (RA) in liver of vitamin A-deficient mice and rats (P < 0.01). LRAT mRNA and enzymatic activity were well correlated in the same livers of rats treated with exogenous RA (r = 0.895, P < 0.0001), and in a dietary study that encompassed a broad range of vitamin A exposure (r = 0.799, P < 0.0001). Liver total retinol of <100 nmol/g was associated with low LRAT expression (<33% of control).We propose that RA, derived exogenously or from metabolism, serves as an important signal of vitamin A status. The constitutive expression of liver LRAT during retinoid sufficiency would serve to divert retinol into storage pools, while the curtailment of LRAT expression in retinoid deficiency would maintain retinol for secretion and delivery to peripheral tissues.     

Metabolism of retinol and retinoic acid by human liver cytochrome P450IIC8

Liver microsomes obtained from nine subjects were found to metabolize retinol to polar metabolites, including 4-hydroxyretinol. In a reconstituted monooxygenase system containing human liver P450IIC8, retinol was converted to 4-hydroxyretinol and other polar metabolites, with a Km of 0.071 mM and a Vmax of 1.73 nmol/min/nmol P450. Neither P450IIC9 nor P450IIE1, two other purified human P450s, displayed significant retinol hydroxylase activity. Immunoblots performed with a monospecific antibody directed against human P450IIC8 revealed that appreciable amounts of this enzyme were present in human liver microsomes. The same antibody significantly inhibited retinol metabolism in liver microsomes and in the system reconstituted with P450IIC8. The system reconstituted with P450IIC8 also converted retinoic acid to polar metabolites. Thus, this study shows, for the first time, metabolism of two physiologic substrates by a human liver cytochrome P450 related to a group of "constitutive" rodent P450s believed to participate in the metabolism of endogenous compounds. Through its involvement in vitamin A metabolism, P450IIC8 may participate in maintaining the balance between those vitamin A concentrations that promote cellular integrity (and oppose the development of cancer) and those concentrations that cause cellular toxicity.     

The involvement of cytochrome p450 (CYP) 26 in the retinoic acid metabolism of human epidermal keratinocytes

All-trans retinoic acid (RA) levels are controlled by enzymes of the vitamin A metabolism (RDH16, RalDH2, and LRAT) and RA catabolism (CYP26 and CYP2S1). Here, the mRNA expression of these enzymes was investigated in human keratinocytes at different Ca²⁺concentrations and after exposure to RA and CYP26 inhibitors. Cellular differentiation (high Ca²⁺) increased the expression of LRAT, RDH16 and RalDH2, and decreased CYP26B1. RA (1 μM) induced CYP26A1, CYP26B1, CYP2S1, CRABPII and LRAT mRNA. The CYP26 inhibitor talarozole altered CYP26A1 and LRAT mRNA expression in a similar way as RA, increased the cellular accumulation of [³H]RA, and induced a punctate CRABPII staining, also observed after siRNA knock-down of CYP26B1 (but not after RA exposure). Furthermore, CYP26B1 siRNA increased the accumulation of [³H]RA and the CRABPII mRNA, suggesting an augmented retinoid signalling. Thus CYP26B1 appears essential for RA catabolism under physiological conditions, whereas CYP26A1 might play a greater role during RA excess.     

Retinoids and retinoid-metabolic gene expression in mouse adipose tissues

Vitamin A and its analogs (retinoids) regulate adipocyte differentiation. Recent investigations have demonstrated a relationship among retinoids, retinoid-binding-protein 4 (RBP4) synthesized in adipose tissues, and insulin-resistance status. In this study, we measured retinoid levels and analyzed the expression of retinoid homeostatic genes associated with retinol uptake, esterification, oxidation, and catabolism in subcutaneous (Sc) and visceral (Vis) mouse fat tissues. Both Sc and Vis depots were found to contain similar levels of all-trans retinol. A metabolite of retinol with characteristic ultraviolet absorption maxima for 9-cis retinol was observed in these 2 adipose depots, and its level was 2-fold higher in Sc than in Vis tissues. Vis adipose tissue expressed significantly higher levels of RBP4, CRBP1 (intracellular retinol-binding protein 1), RDH10 (retinol dehydrogenase), as well as CYP26A1 and B1 (retinoic acid (RA) hydroxylases). No differences in STRA6 (RBP4 receptor), LRAT (retinol esterification), CRABP1 and 2 (intracellular RA-binding proteins), and RALDH1 (retinal dehydrogenase) mRNA expressions were discerned in both fat depots. RALDH1 was identified as the only RALDH expressed in both Sc and Vis adipose tissues. These results indicate that Vis is more actively involved in retinoid metabolism than Sc adipose tissue.     

Phytanic acid and docosahexaenoic acid increase the metabolism of all-trans-retinoic acid and CYP26 gene expression in intestinal cells.

Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all-trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRalpha) and peroxisome proliferator activating receptor (PPARalpha) activator, also activated PPARdelta, and to a lesser degree PPARgamma, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all-trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all-trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all-trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all-trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all-trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all-trans-RA in intestinal cells.     

Diverse actions of retinoid receptors in cancer prevention and treatment

Retinoids (retinol [vitamin A] and its biologically active metabolites) are essential signaling molecules that control various developmental pathways and influence the proliferation and differentiation of a variety of cell types. The physiological actions of retinoids are mediated primarily by the retinoic acid receptors alpha, beta, and gamma (RARs) and rexinoid receptors alpha, beta, and gamma. Although mutations in RARalpha, via the PML-RARalpha fusion proteins, result in acute promyelocytic leukemia, RARs have generally not been reported to be mutated or part of fusion proteins in carcinomas. However, the retinoid signaling pathway is often compromised in carcinomas. Altered retinol metabolism, including low levels of lecithin:retinol acyl trasferase and retinaldehyde dehydrogenase 2, and higher levels of CYP26A1, has been observed in various tumors. RARbeta(2) expression is also reduced or is absent in many types of cancer. A greater understanding of the molecular mechanisms by which retinoids induce cell differentiation, and in particular stem cell differentiation, is required in order to solve the issue of retinoid resistance in tumors, and thereby to utilize RA and synthetic retinoids more effectively in combination therapies for human cancer.     

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

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

2,3,7,8-tetrachlorodibenzo-p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all-trans-retinoic acid (atRA) formation. Male Sprague-Dawley rats were exposed to a single oral dose of TCDD in a combined dose-response and time-course study, with doses ranging from 0.1 to 100 microg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin-O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time-course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.     

Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA

We examined expression of retinal dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate "vitamin A normal" expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol dehydrogenase isozymes (except stellate cells, for which retinol dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism.     

Retinoic acid hydroxylase (CYP26) is a key enzyme in neuronal differentiation of embryonal carcinoma cells.

Besides nuclear retinoid receptors and cellular retinoid binding proteins also retinoic acid (RA)-synthesizing enzymes (using all-trans-retinal as substrate) and RA-catabolizing enzymes (producing hydroxylated products) may explain the specific effects of retinoids. In the past we have established an active role for 4-hydroxy-RA and 4-oxo-RA, which originally were considered to be inactive retinoids, but in fact are highly active modulators of positional specification in Xenopus development. Here we present evidence for a specific role of hydroxylated RA metabolites in the onset of neuronal differentiation. 4-Hydroxy- and 18-hydroxy-RA are products of the hydroxylation of RA by a novel cytochrome P450 (CYP)-type of enzyme, CYP26, expression of which is rapidly induced by RA. P19 embryonal carcinoma (EC) cell lines stably expressing hCYP26 undergo extensive and rapid neuronal differentiation in monolayer at already low concentrations of RA, while normally P19 cells under these conditions differentiate only in endoderm-like cells. Our results indicate that the effects on growth inhibition and RARbeta transactivation of P19 EC cells are mediated directly by RA, while the onset of neuronal differentiation and the subsequent expression of neuronal markers is mediated by hCYP26 via the conversion of RA to its hydroxylated products.     

A novel cytochrome P450, zebrafish Cyp26D1, is involved in metabolism of all-trans retinoic acid

Retinoid signaling is essential for development of vertebrate embryos, and its action is mainly through retinoic acid (RA) binding to its RA receptors and retinoid-X receptors, while the critical concentration and localization of RA in embryos are determined by the presence and activity of retinal dehydrogenases (for RA synthesis) and cytochrome P450 RAs (Cyp26s) (for degradation of RA). Previously, we identified a novel cyp26 gene (cyp26d1) in zebrafish that is expressed in hindbrain during early development. Using reverse-phase HPLC analyses, we show here that zebrafish Cyp26D1 expressed in 293T cells could metabolize all-trans RA, 9-cis RA, and 13-cis RA, but could not metabolize retinol or retinal. The metabolites of all-trans RA produced by Cyp26D1 were the same as that produced by Cyp26A1, which are mainly 4-hydroxy-all-trans-RA and 4-oxo-all-trans-RA. Performing mRNA microinjection into zebrafish embryos, we demonstrated that overexpression of Cyp26D1 in embryos not only caused the distance between rhombomere 5 and the first somite of the injected embryos to be shorter than control embryos but also resulted in left-right asymmetry of somitogenesis in the injected embryos. These alterations were similar to those caused by the overexpression of cyp26a1 in zebrafish embryos and to that which resulted from treating embryos with 1 microm 4-diethylamino-benzaldehyde (retinal dehydrogenase inhibitor), implying that cyp26d1 can antagonize RA activity in vivo. Together, our in vitro and in vivo results provided direct evidence that zebrafish Cyp26D1 is involved in RA metabolism.     

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.     

Retinol conversion to retinoic acid is impaired in breast cancer cell lines relative to normal cells

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.     

Retinoid metabolism during development of liver cirrhosis

The changes in retinoid metabolism have been documented in liver cirrhosis. However, the dynamic alterations in levels of this vitamin between circulation and liver during development of the liver cirrhosis are not well understood. The aim of this study was to measure retinoids in the liver and circulation in parallel, during and after development of cirrhosis induced by carbon tetrachloride and thioacetamide. Retinoid levels were measured by HPLC. A decrease in retinaldehyde and total retinol, together with an increase in retinoic acid was evident in liver from both carbon tetrachloride or thioacetamide treated rats within a month after initiation of treatment. Activity of enzymes involved in retinoid metabolism such as retinaldehyde oxidase, retinaldehyde dehydrogenase, and retinaldehyde reductase were decreased in the liver. In parallel, levels of retinol and retinaldehyde in the serum were increased while retinoic acid was decreased. This study indicates that during development of cirrhosis, there is reciprocal transfer of retinoid metabolites between the circulation and the liver.