RARβ mRNA Research Articles

Fenretinide-induced apoptosis of Huh-7 hepatocellular carcinoma is retinoic acid receptor β dependent

BackgroundRetinoids are used to treat several types of cancer; however, their effects on liver cancer have not been fully characterized. To investigate the therapeutic potential of retinoids on hepatocellular carcinoma (HCC), the present study evaluates the apoptotic effect of a panel of natural and synthetic retinoids in three human HCC cell lines as well as explores the underlying mechanisms.MethodsApoptosis was determined by caspase-3 cleavage using western blot, DNA double-strand breaks using TUNEL assay, and phosphatidylserine translocation using flow cytometry analysis. Gene expression of nuclear receptors was assessed by real-time PCR. Transactivation assay and chromatin immunoprecipitation (ChIP) were conducted to evaluate the activation of RXRα/RARβ pathway by fenretinide. Knockdown of RARβ mRNA expression was achieved by siRNA transfection.ResultsOur data revealed that fenretinide effectively induces apoptosis in Huh-7 and Hep3B cells. Gene expression analysis of nuclear receptors revealed that the basal and inducibility of retinoic acid receptor β (RARβ) expression positively correlate with the susceptibility of HCC cells to fenretinide treatment. Furthermore, fenretinide transactivates the RXRα/RARβ-mediated pathway and directly increases the transcriptional activity of RARβ. Knockdown of RARβ mRNA expression significantly impairs fenretinide-induced apoptosis in Huh-7 cells.ConclusionOur findings reveal that endogenous expression of retinoids receptor RARβ gene determines the susceptibility of HCC cells to fenretinide-induced apoptosis. Our results also demonstrate fenretinide directly activates RARβ and induces apoptosis in Huh-7 cells in a RARβ-dependent manner. These findings suggest a novel role of RARβ as a tumor suppressor by mediating the signals of certain chemotherapeutic agents.

Histone Deacetylase Inhibitors Regulate Retinoic Acid Receptor β Expression in Neuroblastoma Cells by Both Transcriptional and Posttranscriptional Mechanisms

The retinoic acid receptor beta (RARbeta) is a retinoic acid (RA)-inducible tumor suppressor, which plays an important role in the arrest of neuroblastoma cell growth. Using human neuroblastoma SH-SY5Y cells, we have examined the regulation of RARbeta expression by histone deacetylase inhibitors (HDACi), considered to be promising agents in anticancer therapy. Our results show that HDACi cooperated with RA to increase RARbeta mRNA levels and to activate the RARbeta2 promoter in transient transfection assays. Chromatin immunoprecipitation assays showed that the basal RARbeta2 promoter that contains the RA response element was refractory to acetylation by both HDACi and RA. In addition, HDACi caused a transient increase in acetylation of a downstream RARbeta2 region, even though global histones remain hyperacetylated after a prolonged treatment with the inhibitors. RA potentiated this response and maintained acetylation for a longer period. Despite the cooperation of RA with HDACi to increase transcription of the RARbeta gene, these inhibitors caused a paradoxical reduction of the cellular levels of the RARbeta protein in cells treated with the retinoid. This reduction is secondary to a change in the protein half-life that is decreased by the HDACi due to increased ubiquitin-independent proteasomal degradation. These results show that HDACi regulate expression of the tumor suppressor gene RARbeta by both transcriptional and posttranscriptional mechanisms and might then modulate sensitivity to the retinoid in neuroblastoma cells.

Sequence analysis of retinoic acid receptor α, β and γ isoforms in the lizard, Podarcis sicula

Vitamin A and its principal biologically active derivative, retinoic acid (RA), play a fundamental role in diverse processes, such as proliferation, differentiation, morphogenesis, metabolism and apoptosis of many types of cells. In addition, RA has been shown to be involved in the regulation of testicular function. These effects are mediated by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), each with three subtypes α, β and γ. The physiological involvement of retinoids in testicular function has been conducted mainly in mammals. Recently, we found that exogenous all- trans-retinoic acid impairs spermatogenesis and enhance testicular germ cell apoptosis in the lizard, Podarcis sicula, a seasonal breeder. To further investigate the role of retinoic acid in lizard, we focus this work principally on the characterization of lizard retinoic acid receptors (α, β and γ isoforms). RARα is 2720 bp long with a putative ORF between 699 and 2133. A Kozac sequence is present at 696 and a putative poly-adenilation site is present in position 2612. The RARα sequence shares 87% homology with mouse RARα mRNA while it has 76 and 80% homology with lizard RARβ and γ mRNAs. RARβ is 2478 bp long showing a putative ORF between 196 and 1543. A canonical Kozac sequence is present at 193 and a putative poly-adenilation site is present at 2294. RARβ shares 91% homology with mouse RARβ mRNA and has 76% homology with both RARα and γ. RARγ is 2416 bp long. With a putative ORF between 444 and 1818. A Kozac sequence is present at 441 and a putative poly-adenilation site is present at 2288. RARγ shares 86% homology with mouse RARβ mRNA and has 80 and 76% homology with both RARα and β respectively. It is worth to note that, as in mouse, the 5′UTR of all isoforms is TATA and CAAT less. Both Northern blot and PCR analyses indicate that lizard testis expresses only RARα and RARβ mRNAs, while RARγ mRNA transcript was not found. In the period analysed, RARβ was expressed during the gonadal full activity (May) and RARα was present in the post-reproductive period (August). During the autumnal recrudescence (October) RARα and RARβ are co-expressed and, as indicated by quantitative PCR analysis, RARβ mRNA levels are lower than RARα ones. Thus, the appearance and abundance of each receptor correspond to a specific phase of lizard reproductive cycle, allow us to hypothesize that each RAR subtype could play a specific role in the regulation of spermatogenetic activity. The results of the present study show, for the first time, the characterization of RAR mRNAs in the testis of lizard P. sicula, whose expression is related to the different phase of reproductive cycle. Moreover, the γ form, is principally expressed in the skin during the March–July period, having probably a role in regulating skin homeostasis and colour livery, which are important factor in mating during the reproductive cycle.

Expression, protein stability and transcriptional activity of retinoic acid receptors are affected by microtubules interfering agents and all- trans-retinoic acid in primary rat hepatocytes

Cellular signaling by glucocorticoid receptor and aryl hydrocarbon receptor is restricted by microtubules interfering agents (MIAs). This leads to down-regulation of drug metabolizing enzymes and drug interactions. Here we investigated the effects of all- trans-retinoic acid (ATRA) and MIAs, i.e. colchicine, nocodazole and taxol on the regulation of retinoic acid receptor (RAR) genes in primary cultures of rat hepatocytes. ATRA (1 μM) down-regulated RARα and RARγ mRNAs (decrease 23% and 41%, respectively) whereas it up-regulated RARβ mRNA (4.3-fold induction). All MIAs diminished the expression of RARs in dose-dependent manner; the potency of MIAs increased in order NOC < COL < TAX and the extent of inhibition increased in order RARα < RARγ < RARβ. The levels of RARα protein were decreased by both MIAs and ATRA. The effects of ATRA were reversed by proteasome inhibitor MG-132, implying ligand-dependent RARα degradation. In contrast, the effects of MIAs were proteasome-independent and decrease in RARα protein content was due to RARα gene down-regulation. We monitored transcriptional activity of RARα. For this purpose, we measured catalytic activity of trans-glutaminase—target gene of RARα. trans-Glutaminase activity was increased by ATRA (1.23-fold increase) and decreased by colchicine (decrease 51%). Co-treatment with proteasome inhibitor MG-132 partly reversed inhibitory effect of colchicine, and it further augmented the increase of trans-glutaminase activity by ATRA. We have also observed decrease of RARα protein level and inhibition of RARs mRNAs expression in HeLa cells by MIAs. In conclusion, our data indicate that microtubules play the role in regulation of RARs activity and expression. Our data are the first report on the effects of ATRA and MIAs on RARs regulation in quiescent cells.

The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

9- cis-Retinoic acid inhibition of lung carcinogenesis in the A/J mouse model is accompanied by increased expression of RAR-β but no change in cyclooxygenase-2

9- cis-Retinoic acid (9cRA) binds both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) and has been shown to be a potential chemopreventive agent both in lung cancer cell culture studies and in clinical trials studying former smokers. However, direct evidence of the efficacy of 9cRA against lung tumor development in vivo is lacking. In the present study, we determined whether treatment with 9cRA has the potential to inhibit lung carcinogenesis by upregulating RAR-β and down-regulating COX-2 expression in the A/J mouse lung cancer model. A/J mice ( n=14–15/group) were treated as follows: (1) Control (Sham treated); (2) NNK (100 mg NNK/kg body weight); (3) NNK+9cRA (15 mg/kg diet); and (4) NNK+celecoxib (a COX-2-specific inhibitor, 500 mg/kg diet). Tumor incidence, tumor multiplicity, RAR-β mRNA, COX-2 mRNA, and COX-2 protein levels in lung samples of mice were determined 4 months after carcinogen injection. The results showed that mice receiving 9cRA supplementation had significantly lower tumor multiplicity (48% reduction, P<0.05) and showed a trend toward lower tumor incidence (40% reduction, P=0.078), as compared with the mice given NNK alone. Although, celecoxib treatment resulted in greater declines in tumor incidence and tumor multiplicity (75 and 88%, respectively, P<0.05), the chemoprotective effects of celecoxib were accompanied by increased mortality while 9cRA treatment resulted in no weight-loss associated toxicity or mortality. Supplementation with 9cRA was effective in increasing RAR-β mRNA, but this increase was not accompanied by decreased levels of COX-2 mRNA or protein. These results suggest that 9cRA supplementation may provide protection against lung carcinogenesis and this effect may be mediated in part by 9cRA induction of RAR-β, but not inhibition of COX-2 transcription.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARα did not exhibit significant ontogenetic changes. RARβ mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARα levels. Levels of RXRα and RXRβ mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Independent pathways in the modulation of osteoclastic resorption by intermediates of the mevalonate biosynthetic pathway: The role of the retinoic acid receptor

Geranylgeranyl pyrophosphate (GGPP) and geranylgeraniol (GGOH) are used for the prenylation of GTP binding proteins and can reverse the antiresorptive action of nitrogen-containing bisphosphonates which inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway involved in the formation of GGPP. Previously, in cultures of fetal mouse long bones, we showed that GGOH stimulates osteoclastic bone resorption, but the cellular and molecular mode of action is not known. In cell homogenates, it has been found that GGOH can be metabolized to geranylgeranoic acid (GGA) which, like retinoic acid (RA), is a stimulator of retinoic acid receptor (RAR) expression. For this, we examined the involvement of the RAR in the action of GGOH on bone resorption. We show here that RA, GGOH, GGPP and GGA stimulate osteoclastic bone resorption and that this action is reversed by the RAR antagonist AGN-193109. These findings indicate the functional involvement of the RAR in the action of these polyisoprenoids. Moreover, RA, GGOH and GGA all stimulated RARβ mRNA expression in bone explants. However, in contrast to GGOH and GGPP, GGA was not able to reverse the antiresorptive action of ibandronate, a nitrogen-containing bisphosphonate, suggesting that GGA is not involved in protein prenylation. In conclusion, our studies show that both GGOH and GGPP, independent of protein prenylation, stimulate osteoclastic bone resorption via RAR, probably via metabolism into GGA. Identification of such mechanism can help in the better understanding of the role of this metabolic pathway in the regulation of the activity and survival of osteoclasts.

Increased Retinoic Acid Receptor-β4 Correlates In vivo with Reduced Retinoic Acid Receptor-β2 in Esophageal Squamous Cell Carcinoma

Different retinoic acid receptor-beta (RAR-beta) isoforms seem to have contrasting biological effects in human carcinogenesis. Both in vitro and in vivo data indicate that RAR-beta2 expression is frequently lost or reduced (and transfecting RAR-beta2 suppresses growth and promotes apoptosis) in various cancer cells and tissues, whereas RAR-beta4 expression is increased in several cancer cell lines. To clarify the effects of different RAR-beta isoforms in esophageal carcinogenesis, we used real-time quantitative reverse transcription-PCR to assess in vivo RAR-beta mRNA levels in specimens of normal and malignant human esophageal tissue, comparing these levels with each other and the expressions of other genes. RAR-beta2 mRNA expression was significantly reduced (i.e., lower in cancer than normal tissue) in 67% (18 of 27, P = 0.001) and RAR-beta(4) mRNA was increased in 52% (14 of 27, P = 0.054) of our esophageal cancer cases. The expressions of RAR-beta1, chicken ovalbumin upstream promoter-transcription factor-I (COUP-TFI), COUP-TFII, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA were reduced, whereas epidermal growth factor receptor and cyclin D1 expressions were increased in tumor compared with in normal tissues. Reduced RAR-beta2 expression correlated with increased RAR-beta4 expression (P = 0.002) and with the suppression of COUP-TFI and COUP-TFII (P = 0.050 and 0.023, respectively) in tumor samples. These are the first in vivo expression patterns of RAR-beta2 and RAR-beta4 reported in humans or animals and support the in vitro data on these isoforms and their contrasting biological effects in human carcinogenesis.

124 Peptidomimetic inhibitors of Stat3: structure—activity relationships and cellular activity

Retinoic acid (RA) strongly inhibits proliferation of the estrogen (E2)-dependent human breast cancer cell lines MCF7, T47D, and ZR75-1, but not the E2-independent and E2 receptor (ER)-negative lines MDA-MB231, MDA-MB468, BT20 and Hs578T. The specific sensitivity of the E2-dependent cell lines seems not to be caused by an inhibitory effect of RA on ER functioning since RA inhibited the proliferative response not only to E2 but also to insulin. Furthermore, endogenous RA receptors (RARs) hardly impaired transcriptional activation of an E2 responsive element-tk-CAT reporter construct.RARα mRNA was highly expressed in the RA-responsive lines, but not in the unresponsive lines, except BT20. With the exception of Hs578T, also RARβ mRNA expression was low in the unresponsive lines. While in the dependent lines and Hs578T RA activated RA responsive element-dependent transcriptional activity, this response was very low in MDA-MB231, MDA-MB468, and BT20, suggesting that the RA resistance of these latter three ER-negative lines is due to underexpression of functional RARs. Our results suggest that the loss of functional RARs may be a frequent event, leading to RA unresponsiveness of ER-negative breast cancer cells. This implies that both the steroid and retinoid receptor status of breast tumors may be used to predict a successful treatment with retinoids.

Acyclic retinoid activates retinoic acid receptor β and induces transcriptional activation of p21CIP1 in HepG2 human hepatoma cells

Abstract Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with decreased expression of cell cycle-related molecules. These results, taken together with previous in vitro and clinical studies with ACR, suggest that this agent may be useful in the chemoprevention and therapy of hepatoma and possibly other human malignancies. In the present study, we further examined the molecular effects of ACR on the HepG2 human hepatoma cell line, focusing on the expression of nuclear retinoid receptors and the cell cycle inhibitor protein p21CIP1. Reverse transcription-PCR assays and Western blot analyses indicated that these cells express retinoic acid receptors (RARs) α, β, and γ, retinoid X receptors (RXRs) α and β, and peroxisome proliferator-activated receptors (PPAR) γ mRNA. Treatment with ACR caused a rapid induction within 3 h of RARβ mRNA and the related protein, but there was no significant change in the levels of the mRNA or proteins for RARs α and γ, RXRs α and β, and PPARγ. There was also a rapid increase in p21CIP1 mRNA and protein in HepG2 cells treated with ACR, and this induction occurred via a p53-independent mechanism. In transient transfection reporter assays, we cotransfected the retinoic acid response element-chloramphenicol acetyltransferase (CAT) reporter gene into HepG2 cells together with a RARβ expression vector. RARβ expression markedly stimulated CAT activity (up to about 4-fold) after the addition of ACR. However, CAT activity in the presence of ACR was only about 2-fold higher than that in the absence of ACR, when cells were cotransfected with RARs α and γ or RXRα. These findings suggest that the growth inhibitory effects of ACR are mediated at least in part through RARβ and that both RARβ and p21CIP1 play critical roles in the molecular mechanisms of growth inhibition induced by ACR.

Vitamin A Prevents High Fat Diet-Induced ACF Developmentand Modifies the Pattern of Expression of Peroxisome Proliferator and Retinoic Acid Receptor m-RNA

Some dietary compounds, among them fats, are modulators of colon cancer risk. This study reports the modulating effects of n-6, with or without vitamin A, on promotion of colon preneoplasic lesions induced by 1,2-dimethylhydrazine (DMH) and on the expression of nuclear receptors (PPARγ, RXRα, and RARβ). One group of male Fisher rats was fed a basic diet (5% safflower oil) and two groups were fed a high-fat diet (HFD, 25% safflower oil). Of these, one was supplemented with 200 IU vitamin A for 5 mo. The safflower oil contained polyunsaturated fatty acids, mainly linoleic acid (73%). The data showed an increasing effect of safflower oil-enriched diet on aberrant crypt foci occurrence and multiplicity. This effect was impaired by vitamin A supplementation. In addition, an HFD-related up-regulation of PPARγ and a concomitant down-regulation of RARβ mRNA expression were observed with or without chemical initiation and were prevented by vitamin A. Moreover, when treated with DMH, HFD rats exhibited a dramatically decreased expression of RXRα mRNA (-49%). It was hypothesized that HFD, leading to hyperexpression of PPARγ, would produce an alteration of retinoic acid signaling and, in this way, create a background modulating colon cancer risk.

Retinoic acid receptors and retinoids are up-regulated in the developing and adult rat prostate by neonatal estrogen exposure.

Exposure to estrogens during the neonatal period interrupts rat prostatic development by reducing branching morphogenesis and by blocking epithelial cells from entering a normal differentiation pathway. Upon aging, ventral prostates exhibit extensive hyperplasia, dysplasia, and massive lymphocytic infiltrate, suggesting that neonatal estrogens may predispose the prostate gland to precancerous lesions. Vitamin A (retinol) and their derivatives (retinoic acids) are known key developmental regulators that bind and activate retinoic acid receptors (RARs). To evaluate whether neonatal estrogenization alters the sensitivity of the developing rat prostate to retinoids, RARalpha, -beta, and -gamma cellular localization and protein levels were analyzed over the course of development and into adulthood by immunocytochemistry and Western analysis, whereas mRNA levels were measured using RT-PCR. In addition, intraprostatic retinol and retinoic acid levels were quantitated on d 10 and 90 using HPLC-mass spectroscopy. Male rats were given 25 micro g estradiol benzoate or oil on d 1, 3, and 5 of life, and prostatic complexes were removed on d 6, 10, 15, 30, and 90. The RARs localized to distinct cell populations: RARbeta was expressed within basal epithelial cells, RARalpha was localized to differentiated luminal epithelial cells and smooth muscle cells, and RARgamma was expressed within periductal stromal cells. Over the normal course of development, total protein and mRNA levels for the RARs declined, so that the adult prostate possessed the lowest amounts of RAR. Exposure to estrogens during the neonatal period resulted in an immediate and sustained increase in RARalpha levels and in the number of cells that expressed RARbeta, whereas RARgamma levels were unaffected. Western analysis confirmed that total prostatic RAR protein levels were significantly increased, whereas RT-PCR demonstrated that RARalpha and RARbeta mRNA levels were markedly elevated in response to estrogenic exposure. The total prostatic retinol content was tripled by estrogenic exposure on d 10 and 90, indicating that the ability to retain retinoids within the prostate was permanently increased. Intraprostatic levels of 9-cis- and all-trans-retinoic acid levels were reduced on d 10, whereas 13-cis-retinoic acid levels were increased in response to estrogens. In the adult prostates of rats exposed neonatally to estrogen, total retinoic acid levels were doubled due to significant increases in both 9-cis- and 13-cis-retinoic acids compared with those in control prostates. In summary, levels of specific RARs and their activating ligands are increased in the prostate gland after neonatal estrogenic exposure, and this effect is permanent throughout the life of the animal. Thus, we hypothesize that alterations in morphogenesis as well as dysplasia in the adult prostate may be mediated in part through augmentation of transcriptional signals in the retinoid pathway.

Enhanced apoptosis and radiosensitization by combined 13-CIS-retinoic acid and interferon-α2a; role of RAR-β gene

Purpose: Combined use of 13-cis-retinoic acid (cRA) and interferon-α2a (IFNα) induced significant radiosensitization in human cervical cancer ME-180 cell line, whereas it failed to achieve similar radiation enhancement in HeLa cells. The differential radiosensitization could be from the difference of retinoic acid receptor (RAR) expression because RAR-β was highly expressed in ME-180 cells in contrast to the HeLa cells where RAR-β was not detectable. We examined the role of this gene in mediating radiosensitization by cRA and IFNα, and explored the mechanism of radiation-induced cell killing.Methods and Materials: Human cervical cancer cell lines, ME-180 and HeLa, were treated with cRA and IFNα followed by radiation. Apoptosis and radiosensitization were quantitated by TUNEL assay (in situ DNA nick end labeling) and colony-forming ability of surviving cells. The cells were transfected with bcl-2 gene and RAR-β gene to test the role of these genes in mediating radiosensitization and apoptosis.Results: Synergistic radiosensitization and apoptosis was observed by combined use of cRA and IFNα with radiation in ME-180 cells which express high level of RAR-β mRNA, whereas these were not seen in HeLa cells where RAR-β mRNA is not detectable. Both radiosensitization and apoptosis were abolished by bcl-2 gene in ME-180 cells. RAR-β gene transfection induced similar radiation enhancement and apoptosis in HeLa cells.Conclusion: Apoptosis and radiation response were enhanced in the cells with high level of RAR-β mRNA expression. The RAR-β gene appears to mediate the radiation-induced apoptosis by cRA and IFNα. These findings indicate that presence of RAR-β in the cancer cells could be exploited for patient selection in using these drugs for apoptosis and radiosensitization.

A Retinoic Acid Receptor Antagonist Suppresses Brain Retinoic Acid Receptor Overexpression and Reverses a Working Memory Deficit Induced by Chronic Ethanol Consumption in Mice

Background: Chronic ethanol consumption induces disorders in the biosynthesis of retinoic acid, an active derivative of vitamin A. Recent evidence suggests that an alteration in the retinoic acid signaling pathway leads to impairments in learning and memory in adult mice. We have previously shown that chronic ethanol consumption in mice produces an increased expression of the brain retinoic acid receptor β (RARβ) mRNA. These results prompted us to examine whether suppressing the overexpression of retinoid receptors in alcohol‐treated mice by RAR antagonist administration would reverse their cognitive impairment.Methods: After 10 months of ethanol consumption (12% v/v in drinking water), C57BL/6 mice were submitted to a working memory task in a T‐maze. Then, mice of the control and the ethanol‐treated groups received an RARβ antagonist (CD2665 0.6 mg/kg) for 22 days. The behavioral effect of CD2665 administration was evaluated on a spontaneous alternation task and the neurochemical effect was measured by quantifying the mRNA expression of RARα, RARβ, retinoid X receptor (RXRβ/γ) and tissue transglutaminase (tTG; a retinoic acid‐target gene).Results: Mice submitted to ethanol treatment exhibited a progressive decrease in spontaneous alternation rates over successive trials. Moreover, these mice displayed an increased expression of brain RARβ and RXRβ/γ mRNA, together with an increased level of tTG mRNA and enzymatic activity. The administration of CD2665 to alcohol‐treated mice totally reversed the working memory deficit and suppressed the overexpression of brain RARβ, RXRβ/γ and tTG mRNA, whereas the same treatment in control mice decreased only the RARβ mRNA level without affecting memory performance.Conclusion: These data point to the potential role of the retinoid signaling pathway in memory processes and suggest that the overexpression of brain RARβ and RXRβ/γ could be responsible, at least in part, for some memory impairments observed during chronic ethanol consumption.

Convergence of c AMP, TGF-β and retinoic acid signaling pathways in cells of the embryonic palate

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-β) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-β1, cyclic adenosine 3′, 5′-monophosphate (cAMP) and RA signaling systems. TGF-β1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-β mRNA in MEPM cells, though only TGF-β1 inhibited RA-induced RAR-β protein expression. Forskolin, but not TGF-β1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-β gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-β-responsive, element. Thus, a putative TGF-β Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-β promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.

The Role of Epigenetic Modifications in Retinoic Acid Receptor β2 Gene Expression in Human Prostate Cancers

The retinoic acid receptor (RAR) β gene is a putative tumor suppressor gene on chromosome 3p24, where a high incidence of loss of heterozygosity is detected in many types of tumors. Retinoic acid suppresses cancer cell growth through binding to RARs, especially RARβ, indicating a critical role in mediating anticancer effects. Selective loss or down-regulation of RARβ mRNA and protein has been reported in prostate cancers (PCas), although the mechanisms remain unclear. We investigated the role of epigenetic modification in RARβ2 gene silencing. Aberrant methylation was detected in 11 of 14 (79%) primary PCas, 9 of 10 (90%) hormone-refractory PCas, and 2 of 4 (50%) PCa cell lines, but not in any normal prostate samples. Chromatin immunoprecipitation assay showed that all RARβ2-negative cells (LNCaP, PC3, and DU145) were hypoacetylated at both histones H3 and H4. After exposure to 5-aza-2prime;-deoxycytidine treatment, Trichostatin A and all-trans retinoic acid induced partial demethylation, increased accumulation of acetylated histones, and markedly restored the expression of RARβ2 in RARβ2-negative cells. These data suggest that the RARβ2 gene may be one of the frequently silenced genes by epigenetic modifications in PCa.

Effect of Benzo[a]pyrene Diol Epoxide on Expression of Retinoic Acid Receptor-β in Immortalized Esophageal Epithelial Cells and Esophageal Cancer Cells

Expression of retinoic acid receptor-beta (RAR-β) is frequently lost in tobacco-related cancers. Benzo[a]pyrene diol epoxide (BPDE) is an active metabolite of tobacco procarcinogen benzo[a]pyrene and plays an important role in tobacco carcinogenesis. We therefore exposed SV-40 immortalized esophageal epithelial cells and esophageal cancer cells to BPDE to understand possible interactions between BPDE and RAR-β expression. Our data showed that BPDE decreased RAR-β mRNA and protein levels by suppression of transcription of RAR-β. Retinoic acid was able to partially block the inhibitory effect of BPDE on RAR-β expression and to increase G1 phase of cell cycles. Furthermore, induction of COX-2 expression by BPDE was associated with RAR-β inhibition. This study suggests that one way by which BPDE causes esophageal carcinogenesis may be through the inhibition of RAR-β.

Existence of Retinoic Acid-Receptor-Independent Retinoid X-Receptor-Dependent Pathway in Myeloid Cell Function

We previously reported that ER-27191 (4-[4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl-1-(3-pyridylmethyl)anthra[1,2-b]pyrrol-3-yl]benzoic acid) is a potent antagonist of retinoic acid receptor (RAR), and ER-35795 ((2E,4E,6E)-7-[1-(1-methylethyl)-8-chloro-1,2,3,4-tetrahydroquinolin-6-yl]-6-fluoro-3-methyl-2,4,6-nonatrienoic acid) is a novel retinoid X receptor (RXR)-specific agonist. By using these compounds, we investigated whether distinct RAR-dependent and RXR-dependent pathways operate to mediate the diverse activities of retinoids, particularly, the effects of the RXR pathway on cellular function. ER-27191 completely antagonized HL60 cell differentiation induced by all-trans’-retinoic acid (atRA). However, the differentiation induced by the ER-35795 was not antagonized at all by the RAR antagonist, but was inhibited by an RXR homodimer antagonist (LGD100754, (2E,4E,6Z)-7-(3-n-propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)-3-methylocta-2,4,6-trienoic acid). Its agonistic action on RXR/RAR heterodimer, on the other hand, was neutralized by the RAR antagonist. During HL60 cell differentiation, atRA induced RARβ mRNA, while the RXR had no effect. Interestingly, a functional RXR-pathway was also seen in lipopolysaccharide-induced inhibition of mouse splenocyte proliferation. These results strongly suggest the existence of a pharmacological RXR-dependent pathway that is activated by a ligand that can bind to RXR.

Retinoic acid receptor-beta as a prognostic indicator in stage I non-small-cell lung cancer.

Retinoids are pivotal in the growth and differentiation of certain epithelial tissues, interacting with nuclear retinoid receptors (the retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which function as transcription factors. RAR-beta mRNA is undetectable by in situ hybridization (ISH) in 50% of non-small-cell lung cancers (NSCLC). RAR-beta may suppress tumorigenicity. Therefore, we hypothesized that loss of expression of RAR-beta gene in stage I NSCLC is a prognostic factor of a poor clinical outcome. We retrospectively analyzed RAR-beta mRNA levels (by ISH using a digoxigenin-labeled antisense riboprobe) in specimens from 185 consecutive patients with completely resected clinical/radiographic stage I NSCLC for whom clinical follow-up data were available. One hundred fifty-six patients who met the criteria of pathologic stage I NSCLC and positivity for RXR-alpha mRNA (used as a control to assess RNA degradation) and who had adequate follow-up could be evaluated. RAR-beta mRNA expression was undetectable in 51 patients, weakly positive in 64 patients, and strongly positive in 41 patients. Overall survival of the 41 patients with strongly positive RAR-beta was significantly worse than for the 115 patients with weak or absent RAR-beta (P =.045). Unexpectedly, strong RAR-beta expression was associated with a significantly worse outcome of early-stage NSCLC. The mechanisms underlying this clinically and biologically important finding should be further explored.