Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD⁺ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Retinol and retinoic acid increase MMP-2 activity by different pathways in cultured Sertoli cells

Diseases such as atherosclerosis, arthritis and cancer have been related with imbalance in ROS production and failures in regulation of the MMPs. Authors suggested a relationship between MPP activity and ROS. Our research group has demonstrated that retinol 7µM induced changes in Sertoli cell metabolism linking retinol treatment and oxidative stress. We verified MMP activity in Sertoli cells treated with vitamin A using gelatin zymography. We found that retinol (7µM) and retinoic acid (1nM) induced MMP-2 activity in Sertoli cells. Antioxidants reversed retinol-induced but not retinoic acid-induced MMP-2 activity. Moreover, retinol but not retinoic acid increased ROS production quantified by DCFH-DA oxidation. We found that retinol and retinoic acid induced ERK1/2 phosphorylation, but only retinol-increased MMP-2 activity was inhibited by UO126, an ERK1/2 phosphorylation inhibitor. Our findings suggested that retinol-induced MMP-2 activity, but not retinoic acid-induced MMP-2 activity, was related to ERK1/2 phosphorylation and ROS production.     

Retinol and retinoic acid increase MMP-2 activity by different pathways in cultured Sertoli cells

Diseases such as atherosclerosis, arthritis and cancer have been related with imbalance in ROS production and failures in regulation of the MMPs. Authors suggested a relationship between MPP activity and ROS. Our research group has demonstrated that retinol 7µM induced changes in Sertoli cell metabolism linking retinol treatment and oxidative stress. We verified MMP activity in Sertoli cells treated with vitamin A using gelatin zymography. We found that retinol (7µM) and retinoic acid (1nM) induced MMP-2 activity in Sertoli cells. Antioxidants reversed retinol-induced but not retinoic acid-induced MMP-2 activity. Moreover, retinol but not retinoic acid increased ROS production quantified by DCFH-DA oxidation. We found that retinol and retinoic acid induced ERK1/2 phosphorylation, but only retinol-increased MMP-2 activity was inhibited by UO126, an ERK1/2 phosphorylation inhibitor. Our findings suggested that retinol-induced MMP-2 activity, but not retinoic acid-induced MMP-2 activity, was related to ERK1/2 phosphorylation and ROS production.     

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.     

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.     

Cell cycle perturbating agents in a line of human thyroid transformed cells in culture (HuT)

Culture and differentiation parameters of a human thyroid transformed cell line (HuT) were analyzed. Treatment with high concentrations of chemical agents namely dimethyl sulphoxide and retinoic acid, exerted a dramatic cytotoxic effect. The exposure of these cells to the lowest doses of retinoic acid as well as to 8 mM–16 mM 3-aminobenzamide a potent inhibitor of poly(ADPribose)polymerase, resulted in a delay of cell proliferation. Poly(ADPribose)polymerase activity was differently affected by retinoic acid (stimulation) and 3-aminobenzamide (inhibition).     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Regulation of Gdnf expression by retinoic acid in Sertoli cells

Glial cell line‐derived neurotrophic factor (GDNF) and retinoic acid (RA) are two molecules crucial for the regulation of the spermatogonial compartment of the testis. During the cycle of the seminiferous epithelium, their relative concentration oscillates with lower GDNF levels in stages where RA levels are high. It has been recently shown that RA negatively regulates Gdnf expression but the mechanisms behind are so far unknown. Here, we show that RA directly downregulates Gdnf mRNA levels in primary murine Sertoli cells through binding of RARα to a novel DR5‐RARE on Gdnf promoter. Pharmacological inhibition and chromatin immunoprecipitation–quantitative polymerase chain reaction analysis suggested that the underlying mechanism involved histone deacetylase activity and epigenetic repression of Gdnf promoter upon RA treatment.     

Dickkopf‐3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells

Dickkopf‐3 (Dkk‐3) and Dkkl‐1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera‐2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk‐3 but not of Dkkl‐1. Ectopic expression of both Dkk‐3 and Dkkl‐1 induced apoptosis in NT2 cells. Gene silencing of Dkk‐3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk‐3‐silenced cells, this morphological response was not observed in Dkk‐3‐silenced cells. These findings suggest that Dkk‐3 plays a role in the regulation of cell interactions during RA‐induced neuronal differentiation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1243–1254, 2014     

The conversion of ethanolamine and of its metabolites to choline in human neuroblastoma clones: Effect of differentiation induced by retinoic acid

The sequential methylation of ethanolamine (Etn) or phosphorylethanolamine to the corresponding choline (Cho) derivatives was studied in both undifferentiated and retinoic acid (RA) differentiated human neuroblastoma clones LA-N-1 and LA-N-2. Conversion of Etn derivatives to the respective Cho metabolites was low in both cell types. However, after treatment of the cultures with ethanol or RA, the methylation of phosphoryl-Etn was stimulated while that of phosphatidyl-Etn was severely reduced in both cholinergic LA-N-2 and catecholaminergic LA-N-1 cells.Abbreviations Etn ethanolamine - MeEtn monomethylethanolamine - Me₂Etn dimethylethanolamine - Cho choline - PEtn phosphorylethanolamine - PtdEtn phosphatidylethanolamine - LPtdEtn lysophosphatidylethanolamine - RA retinoic acid     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Enhanced antibody production of human-human hybridomas by retinoic acid

The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10^-7 M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas.     

Impaired retinol utilization in Adh4 alcohol dehydrogenase mutant mice

Adh4, a member of the mouse alcohol dehydrogenase (ADH) gene family, encodes an enzyme that functions in vitro as a retinol dehydrogenase in the conversion of retinol to retinoic acid, an important developmental signaling molecule. To explore the role of Adh4 in retinoid signaling in vivo, gene targeting was used to create a null mutation at the Adh4 locus. Homozygous Adh4 mutant mice were viable and fertile and demonstrated no obvious defects when maintained on a standard mouse diet. However, when subjected to vitamin A deficiency during gestation, Adh4 mutant mice demonstrated a higher number of stillbirths than did wild‐type mice. The proportion of liveborn second generation vitamin A‐deficient newborn mice was only 15% for Adh4 mutant mice but 49% for wild‐type mice. After retinol administration to vitamin A‐deficient dams in order to rescue embryonic development, Adh4 mutant mice demonstrated a higher resorption rate at stage E12.5 (69%), compared with wild‐type mice (30%). The relative ability of Adh4 mutant and wild‐type mice to metabolize retinol to retinoic acid was measured after administration of a 100‐mg/kg dose of retinol. Whereas kidney retinoic acid levels were below the level of detection in all vehicle‐treated mice (<1 pmol/g), retinol treatment resulted in very high kidney retinoic acid levels in wild‐type mice (273 pmol/g) but 8‐fold lower levels in Adh4 mutant mice (32 pmol/g), indicating a defect in metabolism of retinol to retinoic acid. These findings demonstrate that another retinol dehydrogenase can compensate for a lack of Adh4 when vitamin A is sufficient, but that Adh4 helps optimize retinol utilization under conditions of both retinol deficiency and excess. Dev. Genet. 25:1–10, 1999. © 1999 Wiley‐Liss, Inc.     

All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells

The effect of all-trans retinoic acid (ATRA) on leukaemia cell differentiation, proliferation and induction of apoptosis was studied by using autonomously growing blast cells isolated from eight patients with acute myeloblastic leukaemia (AML) either at diagnosis ( n=4) or at relapse (n=4). No morphological or functional differentiation induced by ATRA was observed in any of the cases studied. In cell cultures, inhibition of leukaemia cell growth by ATRA was obvious, especially in the case of clonogenic cells, and it was both time- and concentration-dependent. Induction of apoptosis was more difficult to achieve. The cells retained over 90% viability in suspension when the ATRA exposure at any of the concentrations studied was 48 h or less. When the time of exposure to ATRA was longer than 48 h, the viability of the cells decreased in a concentration-dependent manner. Apoptosis was observed morphologically in each of the AML cases with 10-5 to 10-8 M ATRA, if the incubation time of cells in ATRA was 72 h. The percentage of apoptotic cells increased with increasing ATRA concentrations from 12± 9% of 10-8 M ATRA to 78±12% of 10-5 M ATRA. The DNA electrophoretic method was able to detect apoptosis in all the AML samples exposed to 10-7 and 10-6 ATRA for 48 h and occasionally in cases where lower concentrations and longer exposure time were used. In conclusion, the present study shows that it is possible to induce apoptotic leukaemia cell death in vitro with ATRA in AML, and this effect is dependent on both concentration and exposure time.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

Retinoic acid reduces migration of human breast cancer cells: role of retinoic acid receptor beta

Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor β (RARβ) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARβ protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARβ gene expression that was greatest after 72 hrs with a concentration 1 μM. High concentrations of RA increased the expression of RARβ causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration‐related proteins [moesin, c‐Src and focal adhesion kinase (FAK)]. The treatment with RARα and RARγ agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARβ‐agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARβ gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin, c‐Src and FAK expressions. RARβ is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration.     

Induction of apoptosis and change of bcl—2 expression in macrophage Ana—1 cells by all—trans retinoic acid

Macrophage cells play an important role in the initiation and regulation of the immune response.All-trans retinoic acid (ATRA) and its natural and synthetic analogs (retinoids)affect a large number of biological processes.Recently,retinoids have been shown promise in the therapy and prevention of various cancers.However,many interesting questions related to the activities of retinoids remain to be answered:(I) Molecular mechanisms by which retinoids exert their effects;（Ⅱ)why the clinical uses of retinoids give undesirable side effects of varying severity with a higher frequency of blood system symptoms;(Ⅲ)little is known for its impacts on macrophage cells etc.We set up this experiment,therefore,to examine the apoptosis of ATRA on macrophage Ana-1 cell line.Apoptosis of the cells was quantitated,after staining cells with propidium iodide(PI),by both accounting nuclear condensation and flow cytometry.When the cells were treated with ATRA at or higher than 1μM for more than 24h,significant amount of the apoptotic cells was observed.Induction of apoptosis of Ana-1 cells by ATRA was in time-and dose-dependent manners,exhibiting the similar pattern as the apoptosis induced by actinomycin D (ACTD).ATRA treatment of Ana-1 cells also caused the changes of the mRNA levels of apoptosis-associated gene bcl-2,as detected by Northern blot analysis.The temporal changes of bcl-2 expression by ATRA was also parallel to that by ACTD.In conclusion,ATRA can induce apoptosis in macrophage cells,which may be helpful in understanding of immunological functions retinoids.