Retinoic Acid Receptor Protein Research Articles

Evening chronotype is associated with elevated biomarkers of cardiometabolic risk in the EpiHealth cohort: a cross-sectional study.

Study ObjectivesIndividuals with evening chronotype have a higher risk of cardiovascular and metabolic disorders, although the underlying mechanisms are not well understood. In a population-based cohort, we aimed to investigate the association between chronotype and 242 circulating proteins from three panels of established or candidate biomarkers of cardiometabolic processes.MethodsIn 2,471 participants (49.7% men, mean age 61.2 ± 8.4 SD years) from the EpiHealth cohort, circulating proteins were analyzed with a multiplex proximity extension technique. Participants self-reported their chronotype on a five-level scale from extreme morning to extreme evening chronotype. With the intermediate chronotype set as the reference, each protein was added as the dependent variable in a series of linear regression models adjusted for confounders. Next, the chronotype coefficients were jointly tested and the resulting p-values adjusted for multiple testing using a false discovery rate (5%). For the associations identified, we then analyzed the marginal effect of each chronotype category.ResultsWe identified 17 proteins associated with chronotype. Evening chronotype was positively associated with proteins previously linked to insulin resistance and cardiovascular risk, namely retinoic acid receptor protein 2, fatty acid-binding protein adipocyte, tissue-type plasminogen activator, and plasminogen activator inhibitor 1 (PAI-1). Additionally, PAI-1 was inversely associated with the extreme morning chronotype.ConclusionsIn this population-based study, proteins previously related to cardiometabolic risk were elevated in the evening chronotypes. These results may guide future research in the relation between chronotype and cardiometabolic disorders.

Expression of proteins related to thyroid hormone function in the uterus is down-regulated at the day of implantation in hypothyroid pregnant rats.

Hypothyroidism has been linked to infertility, but the mechanisms underlying infertility-related hypothyroidism have yet to be fully elucidated. Therefore, in this study, effects of hypothyroidism on expression of the proteins related to thyroid hormone function in the uterus, which were thought to play a role implantation, including thyroid hormone receptor (TR), thyroid stimulating hormone receptor (TSHR), retinoic acid receptor (RAR) and extracellular kinase (ERK) were identified. Pregnant female rats were rendered hypothyroid by giving methimazole (MMI), orally. Following hypothyroid induction, rats were grouped into control (non-treated) and received subcutaneous thyroxine at 20, 40, and 80 μg/kg/day for five consecutive days. At Day 6, which is the day of implantation (GD 6), rats were sacrificed and the number of embryo implantation site in the uterus was calculated. Then, uterine horns were harvested and expression of the above proteins and their mRNAs were identified by Western blotting and real-time PCR, respectively. In non-treated hypothyroid pregnant rats, the number of embryo implantation sites decreased as compared to euthyroid and hypothyroid rats receiving thyroxine treatment. Similarly, expression of TRα-1, TRβ-1, TSHR, ERK1/2 and RAR proteins and mRNA in the uterus of non-treated hypothyroid rats also decreased (P < 0.05 when compared to euthyroid and thyroxine-treated hypothyroid rats). In conclusion, downregulated expression of the thyroid hormone related proteins in the uterus at the day of implantation might result in infertility as reported in hypothyroid condition.

Marginal Vitamin A Deficiency Affects the Expression Levels and Localization of Retinoic Acid Receptor and Retinoid X Receptor in Rats Meibomian Gland

ABSTRACTPurpose: To assess the effects of marginal vitamin A deficiency (MVAD) on rat Meibomian gland morphology as well as retinoic acid receptor (RAR) and retinoid X receptor (RXR) amounts and localization.Methods: Eight adult female rats were randomized into MVAD and vitamin A normal (VAN) groups, and fed vitamin A-deficient and normal control diets, respectively, for 3 weeks before mating. At postnatal week 4 (P4w), eight newborn animals in both groups were euthanized, respectively. Then, serum retinol levels were analyzed. Meibomian glands from P4w animals were further assessed upon hematoxylin and eosin staining. RAR and RXR gene and protein expression amounts were detected by real time polymerase chain reaction (qRT-PCR), immunofluorescence and western blotting, respectively.Results: Secretory cell nuclei had reduced areas, while the multilayered squamous epithelium of the ductule was overtly thinner in MVAD rats compared with VAN animals. RXR-α showed highest mRNA amounts among all RA receptors; compared with control values, RXR-α showed overtly decreased mRNA amounts in animals with MVAD. In addition, compared with normal rats, MVAD rats had starkly lower RXR-α and RXR-γ expression levels. Furthermore, only the RXR-α protein was detected in the nucleus of cells forming the multilayered squamous epithelium of the ductule in VAN rats.Conclusion: RARs and RXRs may be involved in the regulation of Meibomian gland function. RXR-α and 9-cis RA signaling affects Meibomian gland function, and the mechanisms of retinoids on secretory cells and the ductule epithelium may differ.Abbreviations: MVAD: marginal vitamin A deficiency; RAR: retinoic acid receptor; RXR: retinoid X receptor; VAN: vitamin A normal; RA: retinoic acid; RAREs: retinoic acid responsive elements; HPLC: high performance liquid chromatography; atRA: all-trans retinoic acid; MGD: Meibomian gland dysfunction; CRABPs: cellular retinoic acid-binding proteins

Abstract 2477: An orally bioavailable and selective histone deacetylase (HDAC) 1 &amp; 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma

Abstract INTRODUCTION: Neuroblastoma (NB) is an extra-cranial solid cancer and is among the most common cancers in infants less than 1 year of age, with 650 new cases each year in the United States. Half of the children with NB have high risk disease and 20-50% of those will fail to respond adequately to current therapies, illustrating an urgent unmet medical need. Current treatment for high-risk disease is aggressive, including chemotherapy, surgery, radiation with stem cell transplant, anti-GD2/cytokine immunotherapy and retinoic acid (RA) treatment. RA is a pro-differentiation agent that slows growth and promotes cell death. A gene expression pattern associated with RA-induced NB differentiation was identified, and chemical inhibition of HDAC1/2 was shown to induce a similar expression pattern. METHODS: In this work, we examine the activity of an orally bioavailable HDAC1/2 inhibitor (HDAC1/2i) on NB cell differentiation, proliferation and apoptosis. RA combined with HDAC1/2i enhances gene expression patterns associated with differentiation, slows cellular proliferation and more rapidly induces dendrite formation than RA can achieve alone. The mechanisms leading to the differentiated phenotype were examined by RT-PCR, gene expression microarray and retinoic acid receptor (RAR) chromatin immunoprecipitation followed by high-througput sequening (ChIP-Seq). RESULTS: HDAC1/2i and RA together caused increased localization of the RAR to its own RARα and RARβ promoter regions, and increased in RAR mRNA and protein relative to the RA treatment condition alone. Additionally, expression of Cyp26, an enzyme responsible for clearing intercellular RA, was reduced in the combination setting. Gene set enrichment analysis of the microarray data comparing the combination setting against RA as a single agent suggested that the addition of HDAC1/2i enhanced apoptotic pathways and decreased E2F driven cell cycle signaling. We confirmed enhanced apoptosis in the combination setting by measuring caspase 3 and PARP cleavage. Consistent with this finding, we observed reduced proliferation, increased sub-G1 cell frequency in cell cycle assays and ablation of emergent RA-resistant NB colonies after combination treatment. Further, combination treatment reduced the E2F-activators CDK4 and CDK6 at the protein level while the CDK inhibitor, p21, was dramatically increased. Hypo-phosphorylation of retinoblastoma protein, directly linked to E2F complex inactivation, was also observed and consistent with reduced proliferation and the decreased frequency of S-phase cells observed in EDU incorporation assays. Xenograft models of NB with RA and HDAC1/2i are in progress, as are HDAC1, HDAC2, acetylated histone H3K9 ChIP-seq experiments, and will be discussed. Taken together, these findings support a role for selective HDAC1/2i in combination with RA for the treatment of patients with high risk NB. Citation Format: David L. Tamang, Pengyu Huang, Olga Golonzhka, Jeffrey R. Shearstone, Steven N. Quayle, Simon S. Jones, Min Yang. An orally bioavailable and selective histone deacetylase (HDAC) 1 &amp; 2 inhibitor enhances retinoic acid mediated differentiation of neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2477.

Expression of a retinoic acid receptor (RAR)-like protein in the embryonic and adult nervous system of a protostome species

The vitamin A metabolite, retinoic acid, is an important molecule in nervous system development and regeneration in vertebrates. Retinoic acid signaling in vertebrates is mediated by two classes of nuclear receptors, the retinoid X receptors (RXRs) and the retinoic acid receptors (RARs). Recently, evidence has emerged to suggest that many effects of retinoic acid are conserved between vertebrate and invertebrate nervous systems, even though the RARs were previously thought to be a vertebrate innovation and to not exist in non-chordates. We have cloned a full-length putative RAR from the CNS of the mollusc Lymnaea stagnalis (LymRAR). Immunoreactivity for the RAR protein was found in axons of adult neurons in the central nervous system and in growth cones of regenerating neurons in vitro. A vertebrate RAR antagonist blocked growth cone turning induced by exogenous all-trans retinoic acid, possibly suggesting a role for this receptor in axon guidance. We also provide immunostaining evidence for the presence of RAR protein in the developing, embryonic CNS, where it is also found in axonal processes. Using qPCR, we determined that LymRAR mRNA is detectable in the early veliger stage embryo and that mRNA levels increase significantly during embryonic development. Putative disruption of retinoid signaling in Lymnaea embryos using vertebrate RAR antagonists resulted in abnormal eye and shell development and in some instances completely halted development, resembling the effects of all-trans retinoic acid. This study provides evidence for RAR functioning in a protostome species.

Nuclear factor of activated T-cells 1 increases sensitivity of v-myb transformed monoblasts to all-trans retinoic acid

Nuclear factors of activated T-cells (NFATs) are important regulators of the cytokine gene expression in activated T-cells. In the last decade, NFATs have been shown to regulate cell cycle, differentiation and apoptosis in cells of various origins revealing their importance for cell homeostasis. In this study, we investigated the effects of NFAT1 on proliferation and differentiation of v-myb-transformed BM2 monoblasts. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once overexpressing NFAT1, they became sensitive to all-trans retinoic acid (ATRA). The ATRA-treated BM2NFAT1 cells differentiated along monocyte/macrophage pathway as evidenced by changes in cell morphology, adherence, phagocytic and non-specific esterase activities, reactive oxygen species production, and vimentin expression. Furthermore, overexpressed NFAT1 either alone or in combination with the ATRA-driven signalling pathway deregulated cyclin A and retinoic acid receptor proteins in BM2 cells. Data presented in this study indicate that the NFAT1 and ATRA signalling pathways synergize in control of proliferation and differentiation of BM2 monoblasts.

Retinoic acid receptor and retinoid X receptor subtype expression for the differential diagnosis of thyroid neoplasms

Although differential expression of retinoic acid receptor (RAR) subtypes between benign and malignant thyroid tissues has been described, their diagnostic value has not been reported. To investigate the diagnostic accuracy of RAR and retinoid X receptor (RXR) subtype protein expression for the differential diagnosis of thyroid neoplasms. We used a tissue array containing 93 benign thyroid tissues (normal thyroid, multinodular goiter, and follicular adenoma (FA)) and 77 thyroid carcinomas (papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, and follicular variant of PTC (FVPTC)). Immunostaining was done for RAR and RXR subtypes. Staining was analyzed semiquantitatively based on receiver operating curve analyses and using hierarchical cluster analysis. We found increased expression of cytoplasmic (c) RARA, cRARG, cRXRB and decreased expression of nuclear (n) RARB, nRARG, and nRXRA in thyroid carcinomas compared with benign tissues. We found three proteins differently expressed between FA and FTC and five proteins differentially expressed between FA and FVPTC, with high diagnostic accuracies. Using cluster analysis, the combination of negative staining of membranous RXRB and positive staining for cRXRB had a high positive predictive value (98%) for malignant thyroid disease, whereas the combination of positive nRXRA and negative cRXRB staining had a high predictive value (91%) for benign thyroid lesions. We conclude that differences in RAR and RXR subtype protein expression may be valuable for the differential diagnosis of thyroid neoplasms. The results of this study and especially the value of cluster analysis have to be confirmed in subsequent studies.

The Immunohistochemical Expression of Nuclear Retinoid Receptor and CREB in Gastric Adenocarcinoma

Purpose: Transcriptional factors of CREB (cAMP response element binding protein) are involved in regulating the gene expression in response to a variety of signaling pathways. The proteins produced by the CREB genes play key roles in many physiological processes, including memory and long-term potentiation. The retinoic acid receptor (RAR) axis mediates epithelial cell differentiation and proliferation in many tissues. This study examined the expressions of RAR and CREB and their relationship with the clinicopathologic factors and their significance. Materials and Methods: The levels of the RAR and CREB expressions were measured in 150 gastric adenocarcinomas by performing immunohistochemical staining. Results: 1. An RAR protein expression was found in 63.3% of the adenocarcinomas (95/150) and a CREB expression was found in 60.7% (91/150) of the adenocarcinomas. 2. An RAR protein expression was found in 72.2% (78/108) of the intestinal type adenocarcinomas and in 40.5% (17/42) of the diffuse type adenocarcinomas (P0.05). Based on the depth of invasion, a CREB expression was found in 50% (12/24) of the T1 adenocarcinomas, in 52.4% (11/21) of the T2 adenocarcinomas, in 64.6% (62/96) of the T3 adenocarcinomas, in 66.6% (6/9) of the T4 adenocarcinomas, in 71.1% (59/83) of the adenocarcinomas with lymph node metastasis and in 47.8% (32/67) of the adenocarcinomas without lymph node metastasis (P

Decreased Expression of Retinoid Receptors in Melanoma: Entailment in Tumorigenesis and Prognosis

Retinoids inhibit proliferation and induce differentiation in melanoma cells. Retinoic acid receptors (RAR) and retinoid X receptors (RXR) mediate the various modulatory effects of retinoids in cells. We have studied the in situ expression of each RAR and RXR protein (alpha, beta, gamma) in a large series of melanocytic lesions and correlated the expression with clinicopathologic features and prognosis of the patients. Tissue microarray blocks of 226 melanocytic lesions were semiquantitatively evaluated by immunohistochemistry for the cytoplasmic and nuclear expression of RAR and RXR protein (alpha, beta, gamma). A significant decrease of RARbeta protein (P < 0.0001), nuclear expression of RARgamma (P < 0.0001), and RXRalpha (P < 0.0001) was found in primary and metastatic melanomas as compared with nevi. Loss of nuclear immunoreactivity for RARgamma (P = 0.048) and RXRalpha (P = 0.001) was observed in the lesions showing vertical growth pattern. In addition, in patients with concomitant loss of cytoplasmic staining for RARalpha and RXRalpha, the probability of overall survival (log-rank test, P = 0.002) and disease-specific survival (log-rank test, P = 0.014) was significantly lower. Aberrant expression of retinoid receptors seems to be a frequent event in melanoma and suggests an impairment of the retinoid pathway in this cancer. Our data indicate the loss of retinoid receptor expression with melanoma progression and suggest a possible prognostic significance of the analysis of retinoid receptors in melanoma.

Omega-3 polyunsaturated fatty acids reverse age-related decreases in retinoic acid receptors, retinoid x receptors and peroxisome-proliferator-activated receptors in rat forebrain

Retinoic acid receptors (RAR), retinoid X receptors (RXR) and peroxisome proliferator-activated receptors (PPAR) are transcription factors involved in many cellular processes including learning and memory. These nuclear receptors can be divided into three isotypes: alpha, beta and gamma for RAR and RXR, and alpha, beta/delta and gamma for PPAR. The levels of mRNA for RARs and RXRs decline with ageing, and this decrease can be reversed by treatment with retinoic acid [1, 2], which is an agonist at these receptors. Treatment with retinoic acid also alleviates agerelated memory defi cits. Omega-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) also act as endogenous ligands at PPAR and RXR. We investigated by western blot analysis of rat forebrain if an age-related decline occurred in RAR, RXR and PPAR protein levels, and whether dietary supplementation with EPA and DHA infl uenced this decline. Twenty-four month-old male Wistar rats were fed a standard diet or a diet supplemented with EPA and DHA for 12 weeks. The average daily intake was 145-160 mg EPA and 95-110 mg DHA/kg rat, with an omega-6/omega-3 ratio of 1, provided by the addition of Maxepa® oil to the diet. The control group received the standard diet, with an omega-6/omega-3 ratio of 14. With ageing there were decreases in the protein levels of RARalpha (18%), RARgamma (14%), RXRalpha (36%), RXRbeta (47%) and PPARgamma (40%). Some of these decreases reached signifi cance (P < 0.05 vs. adult controls). They were fully reversed by supplementation with omega-3 PUFA. These observations support the neuroprotective potential of omega-3 fatty acids in ageing and suggest that reversal of age-related changes in retinoid signalling may be involved in their therapeutic effect.

Regulation of tyrosine hydroxylase gene expression by retinoic acid receptor

Retinoic acid (RA), a derivative of vitamin A, critically controls brain patterning and neurogenesis during embryogenesis, and is known to regulate morphological differentiation of catecholaminergic neuronal cells. In this study, we investigated whether the retinoic acid receptor (RAR), a transcription factor specifically activated by all-trans-RA, could directly regulate transcription of tyrosine hydroxylase (TH), the first and rate-limiting step in the catecholamine biosynthesis pathway. First, treating TH-expressing human neuroblastoma SK-N-BE(2)C cells with all-trans RA resulted in an approximately 1.7-fold increase in endogenous TH mRNA expression, as determined by real-time PCR analysis. Second, when SK-N-BE(2)C cells were transiently co-transfected with the TH promoter-luciferase reporter construct, reporter gene expression was prominently activated by RAR in a ligand-dependent manner. Third, we identified a putative RAR responsive cis-regulatory element at - 1500 to - 1487 bp in the TH upstream promoter region by deletional and site-directed mutational analysis. Finally, we demonstrated that this putative motif directly interacts with RAR protein in a sequence-specific manner by means of an electrophoretic mobility shift assay. Taken together, our results indicate that the TH gene may be a direct downstream target of the RA signaling pathway and that RAR is able to activate TH transcription through interaction with an upstream sequence motif residing at - 1500 to - 1487 bp.

Immunohistochemical Expression of Nuclear Retinoid Receptor and CREB(cAMP Response Element Binding Protein) in Lung Cancers

Background : Transcriptional factors of the CREB(cAMP Response Element Binding Protein) are involved in the regulation of gene expression in response to a variety of signaling pathways. Proteins produced by the CREB genes play key roles in many physiological processes, including memory and long-term potentiation. The retinoic acid receptor (RAR) axis mediates epithelial cell differentiation and proliferation in many tissues including the lung. Material and method : The RAR and CREB expression levels were examined in 60 adenocarcinomas and 60 squamous cell carcinomas of the lung using immunohistochemical staining. Results : 1) RAR protein expression was found in 58.3%(35/60) of adenocarcinomas and 36.7%(22/60) of squamous cell carcinomas(P0.05). 4) CREB expression was found in 61.7%(37/60) of adenocarcinomas and 40%(24/60) of squamous cell carcinomas( P0.05). 7) RAR and CREB expression was found in 68.5% of lung cancers, and there was a significant correlation between them(P

Role of retinoic acid receptors in squamous-cell carcinoma in human esophagus

BackgroundWorldwide, cancer in the esophagus ranks among the 10 most common cancers. Alterations of retinoic acid receptors (e.g. RARα, β, γ, and RXRα, β, γ) expression is considered to play an important role in development of squamous-cell carcinoma (SCC), which is the most common esophageal cancer. Alcohol consumption and smoking, which can alter retinoic acid receptor levels, have been identified as key risk factors in the development of carcinoma in the aero-digestive tract. Therefore, the aim of the present study was to evaluate protein levels of retinoic acid receptors (i.e. RARα, β, γ, and RXRβ) in esophageal SCC and surrounding normal tissue of patients with untreated SCC and controls.MethodsAll study participants completed a questionnaire concerning smoking and alcohol drinking habits as well as anthropometrical parameters. Protein levels of RARα, β, γ, and RXRβ were determined by Western Blot in normal esophageal tissue and tissue obtained from SCC of 21 patients with newly diagnosed esophageal SCC and normal esophageal tissue of 10 controls.ResultsProtein levels of RARγ were significantly lower by ~68% in SCC compared to normal surrounding tissue in patients with SCC that smoked and/or consumed elevated amounts of alcohol. Furthermore, RARα protein levels were significantly lower (~- 45%) in SCC in comparison to normal esophageal mucosa in patients with elevated alcohol intake. When comparing protein levels of retinoic acid receptors between normal tissue of patients with SCC and controls, RARγ protein levels were found to be significantly higher (~2.7-fold) in normal esophageal tissue of SCC patients than in esophageal tissue obtained from controls. No differences were found for RARα, β, and RXRβ protein levels between normal esophageal tissue of patients and that of controls.ConclusionIn conclusion, results of the present study suggest that alterations of retinoic acid receptors protein may contribute in the development of SCC in esophagus and that in some patients life style (e.g. smoking and alcohol consumption) may be a critical component in the alteration of retinoic acid receptor levels in esophagus.

Retinoids: Inducers of Tumor/Growth Suppressors

The process of carcinogenesis involves genomic disasters that result in inactivation or underexpression of tumor/growth suppressor genes and/or overexpression of oncogenes. Decreased levels of tumor suppressors may result from DNA hypermethylation, mutations, translocation, or transcriptional repression. Therefore, logically, one of the most promising chemopreventive and chemotherapeutic targets appears to be the activation of tumor suppressors. The expression of tumor suppressor genes could be activated by reversal of epigenetic gene silencing by using DNA methyltransferase inhibitors and/or histone deacetylase inhibitors (Zelent et al, 2004). Alternatively, the expression of multiple tumor suppressor genes in cancer cells could be induced by ligand-dependent transcription factors, such as nuclear receptors for the active form of vitamin A, all-trans retinoic acid (ATRA) that belong to the superfamily of steroid/thyroid hormone nuclear receptors. Retinoids (ATRA and its synthetic analogs) exert their anti-proliferative and differentiation effects through retinoic acid receptors (RAR) that function as heterodimers with retinoid X receptors (RXR). RXR, a nuclear receptor for 9-cis retinoic acid, is an obligate partner of RAR in mediating retinoid action. Three distinct isotypes of RAR (, and ) and RXR (, and ) are encoded by separate genes. The RXR–RAR heterodimer binds to retinoic acid response elements (RARE) present in the promoter regions of responsive genes. RARE are direct repeat (DR) of 5'-RGKTCA-3' motifs (where R is A or G and K is G or T) separated by either two (DR-2) or five (DR-5) nucleotides (Nagpal and Chandraratna, 1998).

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.

Regulation of Retinoic Acid Receptor α by Protein Kinase C in B16 Mouse Melanoma Cells

We have previously found that retinoic acid stimulates the expression of protein kinase C alpha (PKC) in B16 mouse melanoma cells. Because it has been reported that PKC can phosphorylate retinoic acid receptor (RAR) and alter its function, we determined whether changes in the level and/or activity of PKC could affect the expression or function of the RAR in B16 melanoma. Using in vivo phosphorylation and band shift techniques, we could not demonstrate that altering PKC activity and/or protein level changed the in vivo phosphorylation of RAR alpha. However activation of PKC resulted in increased RAR alpha protein. Increased receptor protein correlated with a phorbol dibutyrate-stimulated increase in receptor activation function-2 (AF-2)-dependent transcriptional activity. Use of enzyme inhibitors and dominant-negative PKCs indicated that enzyme activity was required for elevation in the RAR alpha. The PKC-mediated increase in RAR alpha was due to a 2.5-fold increase in the half-life of this protein. In contrast, the down-regulation of PKC diminished RAR alpha protein half-life and markedly inhibited AF-2-dependent transcriptional activity. The down-regulation of PKC also inhibited the binding of RAR to a retinoic acid response element and the retinoic acid induction of RAR beta expression. These findings suggest that PKC can influence retinoic acid signaling by altering the stability of RAR protein without directly phosphorylating this receptor.

Involvement of all- trans-retinoic acid in the breakdown of retinoic acid receptors α and γ through proteasomes in mcf-7 human breast cancer cells

Most studies have reported an up-regulation of retinoic acid receptor (RAR) mRNA expression by all- trans retinoic acid (RA). We aimed to study the effect of RA on RAR protein levels in MCF-7 human breast cancer cells. Incubation of these cells with 10 −6 M RA induced a rapid breakdown of both RARα and RARγ in spite of the accumulation of their mRNAs. Proteasome specific inhibitors blocked the RA-induced breakdown of RARs. Furthermore, RA enhanced the formation of the complex between RARα and ubiquitin in a concentration- and time-dependent manner, suggesting the involvement of ubiquitin and proteasome in this reaction. Retinoid X receptor α (RXRα) was also decreased, albeit to a lesser extent, in RA-treated cells. Use of synthetic receptor agonists and antagonists clearly showed that the effect of the retinoid on the breakdown of the retinoid receptors is receptor-ligand agonist-dependent and blunted by the antagonist. An electrophoretic mobility shift assay, using nuclear extracts from RA-treated cells, showed that a reduction in complex formation with hormone response elements correlated with the reduction of RAR and RXR protein. These data suggest that RA induces the breakdown of RARs through a process involving ubiquitination and that this phenomenon causes a reduction in the formation of DNA-receptor complexes.

The Effects of RARα and RXRα Proteins on Growth, Viability, and Differentiation of v-[formula omitted]-Transformed Monoblasts

ABSTRACTRetinoids are important agents which regulate differentiation and proliferation processes in various cell types, including cancer cells. Growth arrest and induction of terminal differentiation demonstrate the tumor-suppressive effects of retinoids on leukemic cells. We studied differentiation, proliferation, and death processes in the cell line of v-myb-transformed monoblasts BM2 and their retinoic acid receptor (RAR) α- and retinoid X receptor (RXR) α-expressing derivatives after exposure to four different retinoids: all-trans retinoic acid, 9-cis retinoic acid, TTNPB, and LG1000153. The effects of retinoids on the phenotype of BM2, BM2RAR, and BM2RXR cells were correlated with the transcription activation function of the v-Myb oncoprotein of avian myeloblastosis virus. We found that the efficiency of terminal differentiation of BM2RAR and BM2RXR cells induced by retinoids is indirectly proportional to the v-Myb transcription activation activity. In contrast, the effects of liganded retinoid receptors on growth of BM2 cells are more complex. Activated RAR protein induces growth inhibition of BM2 cells by suppression of v-Myb function. However, liganded RXR protein is less efficient in cell cycle arrest and rather decreases cellular viability. This process can occur in the presence of active v-Myb protein. These results suggest that ligand-activated RARα protein is primarily engaged in control of proliferation and differentiation of v-myb-transformed monoblasts, while activated RXRα protein controls their differentiation and death.

Oncoprotein v-Myb and Retinoic Acid Receptor α Are Mutual Antagonists

ABSTRACT: The process of hematopoiesis is critically dependent on correct interactions of multiple regulatory molecules and transcription factors. We have studied the interactions of the v-Myb and retinoic acid receptor proteins which have opposing effects on hematopoiesis. While v-mybacts as a transforming oncogene preventing differentiation of monoblasts to macrophages, RARα functions as an anti-oncogene arresting the growth of v-myb-transformed cells and allowing their final myeloid differentiation steps to occur. We found that the retinoic acid receptor α inhibits v-Myb transformation by suppressing the expression of v-Myb target genes typified by themim-1 gene. Conversely, v-Myb protein interferes with RARα transactivation function as well as with retinoic acid-induced apoptosis of HL-60 cells. These results demonstrate that retinoic acid receptor and v-Myb proteins act in antagonistic ways and reciprocally modify each other's functions.

Thyroid hormone, vitamin D and retinoid receptor expression and signalling in primary cultures of rat osteoblastic and immortalised osteosarcoma cells.

3,5,3'-Tri-iodothyronine (T3), 1 alpha,25(OH)2-vitamin D3 (D3) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T3R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T3, D3 and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T3R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T3, D3, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T3R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.