Nuclear all-trans retinoic acid receptors

The present study was undertaken to investigate the effects of selenite (Se^IV) and selenate (Se^VI) on the all-trans retinoic acid (RA)-nuclear retinoic acid receptor (RAR) complex formation in rat liver. We also present the data on the in vitro effects of Se^IV on the RARα and the type I iodothyronine 5′-deiodinase gene expression in the GH₄C₁ rat pituitary tumor cells. Se^IV at 1.0 μmol/L was found to reduce (p<0.05) the RA specific binding to RAR in rat liver. Dithiothreitol (DTT), a protective agent for sulfhydryl groups, was found to be slightly effective in protecting the RAR binding properties when affected by Se^IV. Se^VI at 0.1 μmol/L reduced (p<0.05) the RA specific binding to RAR in liver, as well. Seleno-l-methionine (Se-^II) when compared tol-methionine did not exert any inhibitory effect on the formation of the RA-RAR complex. Se^IV (up to 2.5 μmol/L) has no inhibitory effect on GH₄C₁ cell proliferation as well as the prolactin secretion. Se^IV at 1.0 μmol/L significantly decreases the rate of mRNA synthesis and/or degradation of the α form of the RAR and causes the enhancement of the type I iodothyronine 5′-deiodinase gene expression in GH₄C₁ cells. The results based on in vitro experiments suggest that inorganic selenium may affect the RA specific binding to their cognate receptor molecules, and it may reduce expression of the gene encoding the RARα, with the cell vitality and the cell growth remaining unchanged.     

Apoptotic and anti-proliferative effects of all-trans retinoic acid. Adenine nucleotide translocase sensitizes HeLa cells to all-trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.     

Effect of all-trans retinoic acid on the barrier function in human retinal pigment epithelial cells

To investigate the effects of all-trans retinoic acid (atRA) on the barrier function in human retinal pigment epithelial cells, ARPE-19 cells were cultured on the filters as monolayer with atRA being added in the apical side. The change of epithelial permeability was observed from the measurement of transepithelial electrical resistance (TER), permeability assay, and Western Blot analysis. We discovered that atRA promoted the epithelial barrier function in vitro, and its bioavailability regulates the epithelial barrier, which is accompanied by altering expression of tight junctions (TJ)-associated proteins. Our study indicates that atRA provides barrier-positive elements to the RPE cell.     

Pathways of absorption of retinal and retinoic acid in the rat

The chemical and anatomical pathways of absorption of dietary retinal, retinoic acid, and retinol were examined in rats containing lymph, bile, and duodenal cannulae. The experiments were designed to maintain physiological conditions to the greatest possible extent. In each rat an uninterrupted flow of bile into the duodenum was maintained by connecting the duodenal cannula to the bile duct of a second rat. Labeled vitamin A compounds were introduced into the duodenum in very small amounts (7-14 micrograms) in the form of a bile-lipid mixture resembling normal intestinal contents. Under these conditions, most (70-80%) of the radioactivity recovered after the feeding of labeled retinol or retinal was found in the lymph, predominantly in saturated retinyl esters. In contrast, 92-95% of the radioactivity recovered after the feeding of labeled retinoic acid was found in the bile, and was contained in a mixture of polar metabolites, most of them more polar than free retinoic acid. Two-thirds of the small amount of radioactivity found in lymph after retinoic acid-(14)C feeding was in the form of free retinoic acid. The results indicate that under normal conditions the major pathway of retinal absorption involves its reduction to retinol, which is then esterified and transported via the lymphatics in a manner similar to that of dietary retinol. A small proportion of retinal is apparently normally oxidized, and is then transported via the portal vein and excreted in the bile in a manner similar to that of dietary retinoic acid. The relative importance, in quantitative terms, of these two pathways of retinal metabolism can vary, depending on the status of the animal.     

In vitro formation of retinoic acid from retinal in rat liver.

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.     

The biosynthesis of retinoic acid from retinol by rat tissues in vitro

This report shows that a spectrum of vitamin A-dependent tissues can produce retinoic acid by synthesis in situ, indicates that cellular retinol and retinoic acid binding proteins are not obligatory to retinoic acid synthesis, and provides initial characterization of retinoic acid synthesis by rat tissues. Retinoic acid synthesis from retinol was detected in homogenates of rat testes, liver, lung, kidney, and small intestinal mucosa, but not spleen. Zinc did not stimulate the conversion of retinol into retinoic acid by liver homogenates. Retinoic acid synthesis was localized in cytosol of liver and kidney, where its rate of synthesis from retinol was fourfold (liver) and sevenfold (kidney) slower than from retinal. The synthesis of retinoic acid from retinol required NAD and was not supported by NADP. NADH (0.5 mM) reduced retinoic acid synthesis from retinol, supported by NAD (2 mM), by 50-70%, but was fivefold less potent in reducing retinoic acid synthesis from retinal. Dithiothreitol enhanced the conversion of retinol, but not retinal, into retinoic acid. EDTA inhibited the conversion of retinol into retinoic acid slightly (13%, liver; 29%, kidney). A high ethanol concentration (100 mM), relative to retinoid substrate (10 microM), inhibited retinoic acid synthesis from retinol (liver, 54%; kidney, 30%) and from retinal (30%, liver; 9%, kidney). 4'-(9-Acridinylamino)methansulfon-m-anisidine, an inhibitor of aldehyde oxidase, and disulfiram, a sulfhydryl-group crosslinking agent, were potent inhibitors of retinoic acid synthesis at 10 microM or less, and seemed equipotent in liver and kidney. 4-Methylpyrazole, an inhibitor of ethanol metabolism, also inhibited retinoic acid synthesis from retinol, but was less potent than the former two inhibitors, and affected liver to a greater extent than kidney, particularly with retinal as substrate.     

Preparation and characterization of micelles of oligomeric chitosan linked to all-trans retinoic acid

New amphiphilic chitosan derivatives of all trans retinoic acid-chitosan (RA-chitosan) with different molar feeding ratios of all trans retinoic acid (ATRA) were synthesized. The degree of ATRA substitution ranged from 8.72% to 18.78%. The RA-chitosan formed micelles with an average size of 142.14-208.4 nm, and zeta potential of +27.25 to 34.48 mV. The critical association concentration (CAC) was found to range from 1.3 × 10⁻² to 2.13 × 10⁻² mg ml⁻¹. Upon evaluation, the RA-chitosan shows no significant cytotoxicity on Hela and HepG2 cells. Analysis of micelles loaded with ATRA revealed that the size of micelles decreased by increasing loaded drug content while zeta potential did not change. ATRA was released slowly over 3-day period, and drug content had no effect on the release rate. These phenomena make RA-chitosan micelles as a candidate for drug carrier.     

Effects of dietary retinoic acid on cellular retinol- and retinoic acid-binding protein levels in various rat tissues

A study was conducted to explore the effects of retinoic acid, fed to retinol-deficient rats, on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). Sensitive and specific radioimmunoassays were employed to measure the levels of both CRBP and CRABP. Two groups of six male rats each were fed a purified retinoid-deficient diet supplemented with either: i) retinyl acetate (control group); or ii) retinoic acid (30 mg/kg diet) (retinol deficient-retinoic acid group). The retinoic acid supplementation was begun after 38 days on the retinoid-deficient diet alone, and was continued for 52-54 days. Analysis of the data indicated that only the CRBP level of the proximal epididymis in the retinol-deficient/retinoic acid group differed significantly from (was lower than) the corresponding control level, at the 1% confidence level. CRABP tissue levels did not differ significantly between the two groups. Thus, a moderately large intake of retinoic acid, as the only source of retinoids, had very little effect on the tissue distribution or levels of either its own cellular binding protein (CRABP) or of CRBP. This study provides further information showing that the tissue levels of the cellular retinoid-binding proteins are highly regulated and maintained in rats, even in the presence of marked changes in retinoid nutritional status.     

Anti-tumor effects of all-trans retinoic acid are enhanced by genistein

The effects of all-trans retinoic acid (ATRA) on cancer are complex. ATRA has anti-cancer effects as it promotes cancer cell differentiation. However, ATRA also up-regulates expression of vascular endothelial growth factor (VEGF) in cancer cells, which leads to angiogenesis and can, thus, facilitate cancer growth. Genistein, a crucial non-nutrient component in soybean, exhibits anti-cancer effects by inhibiting protein tyrosine kinase that is involved in up-regulation of VEGF. We hypothesized that genistein, applied simultaneously with ATRA, would counter its undesired angiogenic effects and, thus, enhance the anti-cancer effects of ATRA. The purpose of this study was to document potential synergistic effects of genistein and ATRA in A549 lung adenocarcinoma cells. We further explored the role of genistein on countering the ATRA-induced VEGF expression. We demonstrate that genistein enhances the ATRA-induced growth inhibition of A549 cells by promoting apoptosis. Further, the combined use of ATRA and genistein leads to cancer cell arrest in G0/G1 and G2/M cell cycle phases. Finally, expression of VEGF (both mRNA and protein) was diminished in A549 cells exposed to both ATRA and genistein. In conclusion, our results demonstrate that genistein effectively enhances anti-cancer effects of ATRA, particularly, by countering the ATRA-induced up-regulation of VEGF. Our study provides an experimental basis for combined use of ATRA and genistein in the treatment of lung cancer.     

Effects of all-trans retinoic acid on UVB-irradiated human skin substitute

Retinoids are frequently used for treatment of photodamaged skin. We wished to find out whether photodamage could be attenuated by applying all-trans retinoic acid (RA) during repetitive irradiation. For this purpose, we used human cutaneous cells and tissue: pure monolayer cultures containing either keratinocytes or fibroblasts, and human skin substitute (SS) containing both cell types. All cultures were exposed to 8 mJ/cm² of UVB and were immediately treated with RA (0, 1.5, or 3 μM). The irradiation and RA treatment protocol was repeated until the cells of the nonirradiated culture had reached confluence. In the irradiated SS, RA preserved the structure (epidermal stratification and differentiation) and ultrastructure (well-organized intermediate filaments and desmosomes) in a state comparable to that observed in nonirradiated SS. As well RA maintained secretion of basement membrane components (laminin and type-IV collagen). Following irradiation, cutaneous cells also displayed more proliferative capacity when SS was treated. In the irradiated monolayer cultures, RA maintained the proliferative capacity of fibroblasts and decreased their differentiation whereas the opposite effect was seen on keratinocytes. In conclusion, RA clearly helps protect human skin against photodamage induced by repeated exposure to UVB. J. Cell. Physiol. 181:14–23, 1999. © 1999 Wiley-Liss, Inc.     

Retinol oxidation to retinoic acid in human thyroid glandular cells

Abstract

Retinoic acid is regarded as the retinol metabolite that controls proliferation and differentiation of epithelial cells. In the present study, we investigated the potential role of xanthine dehydrogenase (XDH) in retinoic acid biosynthesis in human thyroid glandular cells (HTGC). In particular, we observed that cellular retinoids binding proteins (CRBPs) are also implicated in the biosynthetic pathway leading to retinoic acid formation in primary cultures of HTGC, as we have already reported for human mammary epithelial cells (HMEC). After partial protein purification, the enzyme responsible for retinoic acid biosynthesis was identified and quantified as XDH by immunoassay, by its ability to oxidize xanthine to uric acid and its sensitivity to the inhibitory effect of oxypurinol. The evidence of XDH-driven formation of retinoic acid in HTGC cultures further corroborates the potential role of XDH in retinoic acid biosynthesis in the epithelia.     

Lack of response to all-trans retinoic acid supplementation in adult dogs following left pneumonectomy.

We showed previously that removing 55-58% of the lung by right pneumonectomy (R-PNX) in adult dogs triggers compensatory growth of the remaining lung, but removing 42-45% of the lung by left PNX (L-PNX) does not. We also showed that, following R-PNX, supplemental all-trans retinoic acid (RA) selectively enhances alveolar capillary endothelial cell volume (Yan X, Bellotto DJ, Foster DJ, Johnson RL, Jr., Hagler HH, Estrera AS, and Hsia CC. J Appl Physiol 96: 1080-1089, 2004). We hypothesized that RA supplementation might enhance compensation following L-PNX and tested this hypothesis by administering RA (2 mg.kg(-1).day(-1), 4 days/wk) or placebo orally to litter-matched adult foxhounds for 4 mo following L-PNX. Resting lung function was measured under anesthesia. Air and tissue volumes of the remaining lung were assessed by high-resolution computed tomography scan and by detailed postmortem morphometric analysis of the fixed lung. There was no significant difference in resting lung function, lung volume, alveolar structure, or septal ultrastructure between RA and placebo treatment groups. We conclude that RA supplementation does not induce post-PNX compensatory lung growth in the absence of existing cellular growth activities initiated by other primary signals.     

In vitro induction of mucosa-type dendritic cells by all-trans retinoic acid

Efficient induction of mucosal immunity usually employs nasal or oral vaccination while parenteral immunization generally is ineffective at generating mucosal immune responses. This relates to the unique ability of resident mucosal dendritic cells (DC) to induce IgA switching and to imprint mucosa-specific homing receptors on lymphocytes. Based on the well-established plasticity of the DC system, this study sought to investigate whether peripheral DC could be modulated toward "mucosa-type" DC by treatment with immunomodulatory, and therefore potentially adjuvant-like, factors. In this study, we show that monocyte-derived DCs pretreated with the vitamin A derivative all-trans retinoic acid (RA) indeed acquired several attributes characteristic of mucosal DC: secretion of TGF-beta and IL-6 and the capacity to augment mucosal homing receptor expression and IgA responses in cocultured lymphocytes. Addition of a TGF-beta-neutralizing Ab to cocultures significantly inhibited alpha4beta7 integrin, but not CCR9 mRNA expression by the lymphocytes. Both alpha4beta7 integrin and CCR9 mRNA expression, but not IgA production, were suppressed in the presence of a RA receptor antagonist. None of the observed effects on the lymphocytes were influenced by citral, a retinal dehydrogenase inhibitor, arguing against a role for de novo-synthesized RA. Collectively, our findings identified a novel role for RA as a mucosal immune modulator targeting DC. Our results further demonstrate that DC can act as efficient carriers of RA at least in vitro. Consequently, RA targeting of DC shows potential for promoting vaccine-induced mucosal immune responses via a parenteral route of immunization.