In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.     

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.     

Metabolism of a physiological amount of all-trans-retinol in the vitamin A-deficient rat

Because only retinol and not all-trans-retinoic acid (atRA) can satisfy all of the functions of vitamin A, we have investigated the retinol metabolites in tissues of vitamin A-deficient (VAD) rats responding to a radioactive dose of [20-(3)H]all-trans-retinol. As expected, atRA is the major vitamin A metabolite present in the target tissues of VAD rats given a physiological dose (1 microg) of [20-(3)H]all-trans-retinol (atROL). Both atROL and atRA were detected by high-performance liquid chromatographic (HPLC) analysis of the radioactivity extracted from the liver, kidney, small intestine, lung, spleen, bone, skin, or testis of these animals. Novel retinol metabolites were observed in the aqueous extracts from the testis, lung, and skin. However, these metabolites were detected in very small amounts and were not characterized further. Importantly, neither 9-cis-retinoic acid (9cRA), 9-cis-retinol (9cROL), nor 13-cis-retinoic acid (13cRA) was present in detectable amounts. The amounts of atRA varied in each tissue, ranging from 0.29 +/- 0.05 fmol of RA/g of tissue in the femurs to 12.9 +/- 4.3 fmol of RA/g of tissue in the kidneys. The absence of 9cRA in vivo was not due to degradation of this retinoid during the extraction procedure or HPLC analysis of the extracted radioactivity. As atROL completely fulfills all of the physiological roles of vitamin A, and 9cRA is not detected in any of the tissues analyzed, these results suggest that 9cRA may have no physiological relevance in the rat.     

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.     

Characterization of a new endogenous vitamin A metabolite

Here, we describe the discovery of a new major endogenous vitamin A metabolite with particularly high hepatic concentrations. This metabolite was isolated from mouse livers and was characterized as 9-cis-4-oxo-13,14-dihydro-retinoic acid (RA) based on mass spectral, ultraviolet, and nuclear magnetic resonance analyses. It was also detected in one human liver. To gain further insight into endogenous retinoid metabolism, mice were fed over a period of 14 days ad libitum with diets enriched with different amounts of retinyl palmitate [15,000, 45,000 or 150,000 international units (IU)/kg diet]. Higher retinyl palmitate amounts in the diet resulted surprisingly in a dose-dependent decrease in all-trans-RA levels in serum, kidney, and brain, whereas levels of 9-cis-4-oxo-13,14-dihydro-RA, retinol, and retinyl esters were dose-dependently elevated in serum, kidney, and liver. 13-cis-RA levels could be detected in serum, liver, and kidney, but were unaffected by the dietary vitamin A status. 9-cis-RA levels were below the detection limit of 0.2 ng/ml serum or 0.4 ng/g tissue. This study indicates that the oxidation at C4 of the cyclohexenyl ring, isomerization of the C9/C10 double bond, and reduction of the C13/C14 double bond are major endogenous metabolic pathways of vitamin A.     

Embryonal carcinoma cell lines stably transfected with mRARbeta2-lacZ: sensitive system for measuring levels of active retinoids.

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.     

Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells.

In the present paper we demonstrate that retinol-retinol-binding protein and chylomicron remnant retinyl esters in concentrations normally found in human plasma inhibit growth of normal human B lymphocytes. Physiological concentrations of retinoic acid (about 30 nM) were less active than physiological concentrations of retinol (about 3 microM). Pharmacological concentrations of retinol and retinoic acid were more active than the concentrations normally found in plasma. Retinol (3 microM) inhibited anti-IgM-mediated DNA synthesis as measured by [3H]thymidine uptake at 72 h by 78%. Furthermore, we found that the cells were blocked in the mid-G1 phase of the cell cycle. Thus, neither MYC up-regulation measured at 3 h nor the expression of the early activation antigen 4F2 was reduced by retinol, whereas the late activation markers (transferrin receptor expression and actinomycin D staining at 48 h of stimulation) were markedly inhibited. Retinol reduced the interleukin 6 production induced by anti-IgM and interleukin 4 after 48 h, whereas the induction of interleukin 6 and tumor necrosis factor by O-tetradecanoylphorbol-13-acetate and ionomycin was less affected. We also noted that the retinoids reduced the formation of plaque-forming cells (i.e. Ig synthesis). These data imply that vitamin A present in human plasma is a normal modulator of B cell function.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Developmental changes in endogenous retinoids during pregnancy and embryogenesis in the mouse.

Vitamin A and its analogs (retinoids) have acquired particular significance in embryonic development since the discovery that retinoic acid (RA) possesses properties of an endogenous morphogen and that embryonic tissues contain specific nuclear receptors for RA. Since the mammalian embryo does not synthesize RA de novo but rather must acquire it directly or in a precursor form from the maternal circulation, we sought to establish the relationship between levels of RA, retinol, and retinyl esters in the maternal system and their acquisition by the embryo, particularly during organogenesis in the mouse. Results indicate profound changes in maternal vitamin A levels during pregnancy in the mouse. These changes were characterized by a large, transient decrease in plasma retinol levels coincident with the period of organogenesis (e.g. gestational Days 9-14), and an apparent increase in mobilization from hepatic stores to the conceptus. During organogenesis, the embryo exhibited a steady increase in retinol levels with little increase in retinyl esters and virtually no change in RA. Analysis of retinoid accumulation patterns in the embryonic liver indicate that functional onset of vitamin A storage occurs by mid-organogenesis. In contrast, placental levels of these retinoids remained unchanged throughout organogenesis. Analysis of the conceptus as a developmental unit revealed that during early organogenesis the majority of retinoids are contained in the placenta (8-fold more than in the embryo). However, by mid-organogenesis the retinoid content of the embryo exceeds that of the placenta. Together, these results provide evidence that pregnancy in the mouse is accompanied by pronounced alterations in maternal retinoid homeostasis that occur coincident with the period of high embryonic sensitivity to exogenous retinoids.     

Retinoic acid isomers produce malformations in postembryonic development of the Japanese flounder,Paralichthys olivaceus

We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.     

Embryonal Carcinoma Cell Lines Stably Transfected with mRARβ2-lacZ: Sensitive System for Measuring Levels of Active Retinoids

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARβ2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC₅₀ values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1–7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed β-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARβ2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC₅₀ value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.     

Endogenous retinoids in the hair follicle and sebaceous gland

Vitamin A and its derivatives (retinoids) are critically important in the development and maintenance of multiple epithelial tissues, including skin, hair, and sebaceous glands, as shown by the detrimental effects of either vitamin A deficiency or toxicity. Thus, precise levels of retinoic acid (RA, active metabolite) are needed. These precise levels of RA are achieved by regulating several steps in the conversion of dietary vitamin A (retinol) to RA and RA catabolism. This review discusses the localization of RA synthesis to specific sites within the hair follicle and sebaceous gland, including their stem cells, during both homeostasis and disease states. It also discusses what is known about the specific roles of RA within the hair follicle and sebaceous gland. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

Either chick embryo dermis or retinoid-treated mouse dermis can initiate glandular morphogenesis from mammalian epidermal tissue

Excess retinoids can cause developing mouse vibrissa follicles to be transformed into mucous glands in organ culture. The objective was to test the hypothesis that retinoids act in this system by altering morphogenetic properties of the dermis. After inititation by retinoic acid (RA) in organ culture, glands were shown to develop further in embryonic skin grafted to the chick chorioallantoic membrane (CAM). Recombinants of 12.5 day mouse epidermis with untreated or RA-treated mouse or chick dermis were then grafted to CAM for 7 days. For homospecific recombinants, 13.5 day mouse dermis originated from 11.5 day skin cultured for 2 days, with or without 5.2 microgram/ml RA. For heterospecific recombinants, 12 day dermis came from chick embryos, previously injected with 250 microgram RA. Glands were absent from the homospecific recombinants including untreated mouse dermis, but appeared in 26% of those with RA-treated dermis. Among heterospecific recombinants, 75% of those with RA-treated chick dermis and 29% of those with untreated dermis had glands. Untreated 10-12 day chick skin contained two forms of endogenous vitamin A, retinol (4.5 microgram/g protein) and dehydroretinol (3.7 microgram/g protein), while 13-14 day mouse skin contained only retinol (1.8 microgram/g protein), as shown by high performance liquid chromatography. RA injection increased retinol and dehydroretinol in chick skin, while RA was undetectable. Thus RA can act through mouse dermis to form epithelial glands and through chick dermis to increase the incidence of glands. The glands in recombinants with untreated chick dermis may result from the higher levels of endogenous retinoids in chick skin, compared with mouse skin.     

Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats.

Retinoic acid (RA), through nuclear retinoid receptors, regulates the expression of numerous genes. However, little is known of the biochemical mechanisms that regulate RA concentration in vivo. CYP26 (P450RAI), a novel cytochrome P450, is expressed during embryonic development, induced by all-trans RA, and capable of catalyzing the oxidation of [3H]RA to polar retinoids including 4-oxo-RA. Here we report that CYP26 expression in adult liver is regulated by all-trans RA and dietary vitamin A, and is correlated with the metabolism of all-trans RA to polar metabolites. In normal mouse and rat liver, CYP26 mRNA was barely detectable; however, after acute treatment with all-trans RA CYP26 mRNA and RA metabolism by liver microsomes were significantly induced. Aqueous-soluble RA metabolites were detected, but their formation was not induced. The expression of retinoid receptors, RAR-gamma and RXR-alpha, was not changed after RA treatment in vivo. In a model of chronic vitamin A ingestion during aging, CYP26 mRNA expression, determined by Northern blot and RT-PCR analysis, increased progressively with dietary vitamin A (P<0.0001; marginal < control < supplemented) and age (P<0.003). The relative expression of CYP26 mRNA was positively correlated with liver total retinol (log10), ranging from undetectable CYP26 expression at liver retinol concentrations below approximately 20 nmol/g to a three- to fourfold elevation at concentrations >10,000 nmol/g (r=0.90, P<0.0001). We conclude that CYP26 expression and RA metabolism are regulated in adult liver not only acutely by RA administration, as may be relevant to retinoid therapy, but under chronic dietary conditions relevant to vitamin A nutrition in humans.     

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.     

Hepatic retinol secretion and storage are altered by dietary CLA: common and distinct actions of CLA c9,t11 and t10,c12 isomers

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid obtained from ruminant products. Previous studies in rats and pigs showed that a dietary equimolar mixture of c9,t11 and t10,c12 CLA isomers induces changes in serum and tissue levels of retinoids (vitamin A derivatives). However, the mechanism(s) responsible for these actions remain(s) unexplored. Given the numerous crucial biological functions regulated by retinoids, it is key to establish whether the perturbations in retinoid metabolism induced by dietary CLA mediate some of the beneficial effects associated with intake of this fatty acid or, rather, have adverse consequences on health. To address this important biological question, we began to explore the mechanisms through which dietary CLA alters retinoid metabolism. By using enriched preparations of CLA c9,t11 or CLA t10,c12, we uncoupled the effects of these two CLA isomers on retinoid metabolism. Specifically, we show that both isomers induce hepatic retinyl ester accumulation. However, only CLA t10,c12 enhances hepatic retinol secretion, resulting in increased serum levels of retinol and its specific carrier, retinol-binding protein (RBP). Dietary CLA t10,c12 also redistributes retinoids from the hepatic stores toward the adipose tissue and possibly stimulates hepatic retinoid oxidation. Using mice lacking RBP, we also demonstrate that this key protein in retinoid metabolism mediates hepatic retinol secretion and its redistribution toward fat tissue induced by CLA t10,c12 supplementation.     

Multiple retinol and retinal dehydrogenases catalyze all-trans-retinoic acid biosynthesis in astrocytes

All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ～40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.