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Introduction: Prostate cancer has emerged as a widespread health concern, with systemic inflammation believed to substantially contribute to its development and progression. The presence of systemic inflammatory responses has been established as an independent predictor of unfavorable long-term outcomes in prostate cancer patients. The goal of this study is to inhibit RXRα and RXRβ receptors, which are involved in prostate cancer, with Luteolin, Formononetin, and Kaempferol, with varying success., Methods: Retinoid X receptors (RXRs) hold crucial roles within the nuclear receptor (NR) superfamily, and compelling evidence from preclinical studies underscores the therapeutic potential of targeting RXRs for treating neurodegenerative and inflammatory conditions. Consequently, the ability to regulate and modulate RXRs using phytoestrogen ligands, Formononetin, Kaempferol, and Luteolin, assume paramount importance in treatment strategies., Results: The comprehensive in silico findings of this study vividly demonstrate the remarkable efficacy of Luteolin in inhibiting and modulating RXRα and RXRβ, while Formononetin emerges as a notably potent suppressor of RXRβ. Kaempferol, as the third compound, also exhibits commendable inhibitory attributes, although its impact is slightly less pronounced compared to the other two., Discussion: These findings highlight the notable binding and inhibition capabilities to RXRα and RXRβ, offering valuable insights for potential prostate cancer treatment avenues warranting further exploration through in vitro and in vivo analyses.

Rationale : Glycogen synthase kinase-3β (GSK-3β) plays key roles in metabolism and many cellular processes. It was recently demonstrated that overexpression of GSK-3β can confer tumor growth. However, the expression and function of GSK-3β in hepatocellular carcinoma (HCC) remain largely unexplored. This study is aimed at investigating the role and therapeutic target value of GSK-3β in HCC. Methods : We firstly clarified the expression of GSK-3β in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we studied whether GSK-3β could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3β in sorafenib treatment response was determined. Co-immunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to explore the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments. Results : We demonstrated that GSK-3β is highly expressed in HCC and associated with shorter overall survival (OS). Overexpression of GSK-3β confers HCC cell colony formation and xenograft tumor growth. Tumor-associated GSK-3β is correlated with reduced expression of retinoic acid receptor-β (RARβ), which is caused by GSK-3β-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARβ promoter. Overexpression of functional GSK-3β impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3β by tideglusib can potentiate 9- cis -RA enhancement of sorafenib sensitivity (tumor inhibition from 48.3% to 93.4%). Efficient induction of RARβ by tideglusib/9- cis- RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARβ. Conclusions : Our findings demonstrate that GSK-3β is a disruptor of retinoid signalling and a new resistant factor of sorafenib in HCC. Targeting GSK-3β may be a promising strategy for HCC treatment in clinic., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

A large body of evidence suggests that cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT), as well as expression and function of retinoid receptors, are pivotal features of tumor initiation, progression, and chemoresistance. This is also true for pancreatic ductal adenocarcinoma (PDAC), which represents a clinical challenge due to poor prognosis and increasing incidence. Understanding the above features of cancer cells could open new avenues for PDAC treatment strategies. The aim of this study was to investigate the relation between CSCs, EMT, and retinoid receptors in PDAC after treatment with the chemotherapeutic agents - gemcitabine and 5-fluorouracil. First, we demonstrated the difference in the expression levels of CSC and EMT markers and retinoid receptors in the untreated Mia PaCa-2 and Panc1 cells that also differed in the frequency of spontaneous apoptosis and distribution between the cell cycle phases. Chemotherapy reduced the number of cancer cells in the S phase. Gemcitabine and 5-fluorouracil modulated expression of CSC markers, E-cadherin, and RXRβ in Panc1 but not in Mia PaCa-2 cells. We suggest that these effects could be attributed to the difference in the basal levels of expression of the investigated genes. The obtained data could be interesting in the context of future preclinical research.

MicroRNAs (miRNAs) play critical roles in the complex networks of virus-host interactions. Our previous research showed that porcine reproductive and respiratory syndrome virus (PRRSV) infection markedly upregulates miR-c89 expression, suggesting that miR-c89 may play an important role in PRRSV infection. The present study sought to determine the function of miR-c89 and its molecular mechanism during PRRSV infection. Using quantitative reverse transcription PCR (RT-qPCR) verification, we demonstrated that both highly pathogenic PRRSV and low-pathogenic PRRSV infection induced miR-c89 expression. The overexpression of miR-c89 significantly suppressed the replication of a variety of PRRSV strains, regardless of the timing of infection. Further, miR-c89 can directly target the 3'UTR of porcine retinoid X receptor β (RXRB) mRNA in a sequence-specific manner. Knockdown affected RXRB expression, as siRNA can suppress the replication of a variety of PRRSV strains. This work not only provides new insights into PRRSV-cell interactions, but also highlights the potential for the use of miR-c89 in the development of new antiviral strategies to combat PRRSV infection.

Scope: β-Carotene (BC), a substitute for vitamin A, is widely used for its benefits. The present study investigates whether in-utero BC administration can alter lipid and glucose homoeostasis in offspring., Methods and Results: Pregnant mice are supplemented with BC (1 mg kg -1 weight) by oral gavage once every 3 days, for a total of six doses. Increased visceral fat may be caused by up-regulated PPARγ (peroxisome proliferator-activated receptor gamma) and RXRα/β (retinoid X receptors) in liver and adipose tissue, and glucose intolerance is observed in F1 adult females prenatally supplemented with BC, while F1 males do not exhibit these symptoms. In females, increased serum leptin, resistin, and IL-6 and reduced adiponectin, caused by visceral obesity, may result in downregulated insulin receptor signaling in muscle and further account for glucose intolerance. Increased pancreatic β-cell mass might compensate for the downregulated insulin gene (ins2). Increased glucagon and α-cell mass, accompanied by upregulated glucagon gene (gcg), might also be risk factors for the development of diabetes., Conclusions: Maternal supplementation with BC disturbs lipid metabolism and induces glucose intolerance in F1 female mice, suggesting that BC supplementation during pregnancy should be used with caution., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hepatic stellate cells are liver-specific perivascular cells, identified as the major source of collagen in liver fibrosis, following their activation and conversion to myofibroblast-like cells. Lycopene is a carotenoid with biological activities and protective effects described in different pathologies, but little is known about its role in liver protection. We evaluated the influence of lycopene on the cell cycle and lipid metabolism and monitored the possible pathways involved in lycopene inhibition of stellate cell activation. Lycopene induced expression of the lipocyte phenotype, with an accumulation of fat droplets in cytoplasm, with high synthesis and turnover of phospholipids and triglycerides. Cell proliferation analysis showed that lycopene reduced the growth of GRX cells. Lycopene induced an arrest in the G0/G1 phase, followed by a decrease of cells in the G2/M phase, regardless of the concentration of lycopene used. Lycopene modulated relevant signaling pathways related to cholesterol metabolism, cellular proliferation, and lipid metabolism. Also, lycopene treatment increased the expression of RXR-α, RXR-β, and PPARγ, important biomarkers of liver regeneration. These results show that lycopene was able to negatively modulate events related to the activation of hepatic stellate cells through mechanisms that involve changes in expression of cellular lipid metabolism factors, and suggest that this compound might provide a novel pharmacological approach for the prevention and treatment of fibrotic liver diseases.

Retinoic acid (RA) promotes differentiation in multiple neurogenic cell types by promoting gene reprogramming through retinoid receptors and also by inducing cytosolic signaling events. The nuclear RXR receptors are one of the main mediators of RA cellular effects, classically by joining the direct receptors of RA, the nuclear RAR receptors, in RAR/RXR dimers which act as transcription factors. Distinct RXR genes lead to RXRα, RXRβ and RXRγ subtypes, but their specific roles in neuronal differentiation remain unclear. We firstly investigated both RXRs and RARs expression profiles during RA-mediated neuronal differentiation of human neuroblastoma cell line SH-SY5Y, and found varying levels of retinoid receptors transcript and protein contents along the process. In order to understand the roles of the expression of distinct RXR subtypes to RA signal transduction, we performed siRNA-mediated silencing of RXRα and RXRβ during the first stages of SH-SY5Y differentiation. Our results showed that RXRα is required for RA-induced neuronal differentiation of SH-SY5Y cells, since its silencing compromised cell cycle arrest and prevented the upregulation of neuronal markers and the adoption of neuronal morphology. Besides, silencing of RXRα affected the phosphorylation of ERK1/2. By contrast, silencing of RXRβ improved neurite extension and led to increased expression of tau and synaptophysin, suggesting that RXRβ may negatively regulate neuronal parameters related to neurite outgrowth and function. Our results indicate distinct functions for RXR subtypes during RA-dependent neuronal differentiation and reveal new perspectives for studying such receptors as clinical targets in therapies aiming at restoring neuronal function., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Multiple myeloma (MM) is an incurable hematologic malignancy due to inevitable relapse and chemoresistance development. Our preliminary data show that MM cells express high levels of PGC1β and LDHA. In this study, we investigated the mechanism behind PGC1β-mediated LDHA expression and its contribution to tumorigenesis, to aid in the development of novel therapeutic approaches for MM. Real-time PCR and western blotting were first used to evaluate gene expression of PGC1β and LDHA in different MM cells, and then, luciferase reporter assay, chromatin immunoprecipitation, LDHA deletion report vectors, and siRNA techniques were used to investigate the mechanism underlying PGC1β-induced LDHA expression. Furthermore, knockdown cell lines and lines stably overexpressing PGC1β or LDHA lentivirus were established to evaluate in vitro glycolysis metabolism, mitochondrial function, reactive oxygen species (ROS) formation, and cell proliferation. In addition, in vivo xenograft tumor development studies were performed to investigate the effect of PGC1β or LDHA expression on tumor growth and mouse survival. We found that PGC1β and LDHA are highly expressed in different MM cells and LDHA is upregulated by PGC1β through the PGC1β/RXRβ axis acting on the LDHA promoter. Overexpression of PGC1β or LDHA significantly potentiated glycolysis metabolism with increased cell proliferation and tumor growth. On the other hand, knockdown of PGC1β or LDHA largely suppressed glycolysis metabolism with increased ROS formation and apoptosis rate, in addition to suppressing tumor growth and enhancing mouse survival. This is the first time the mechanism underlying PGC1β-mediated LDHA expression in multiple myeloma has been identified. We conclude that PGC1β regulates multiple myeloma tumor growth through LDHA-mediated glycolytic metabolism. Targeting the PGC1β/LDHA pathway may be a novel therapeutic strategy for multiple myeloma treatment., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Adrenal gland reportedly expresses many nuclear receptors that are known to heterodimerize with retinoid-X-receptor (RXR) for functions, but the information regarding the glandular RXR is not adequate. Studies of rat adrenal homogenate by Western blotting revealed three RXR proteins: RXRα (55kDa), RXRβ (47kDa) and RXR (56kDa). RXRγ was not detectable. After fractionation, RXRα was almost exclusively localized in the nuclear fraction. In comparison, substantial portions of RXRβ and RXR were found in both nuclear and post-nuclear particle fractions, suggesting genomic and non-genomic functions. Cells immunostained for RXRα were primarily localized in zona fasciculata (ZF) and medulla, although some stained cells were found in zona glomerulosa (ZG) and zona reticularis (ZR). In contrast, cells immunostained for RXRβ were concentrated principally in ZG, although some stained cells were seen in ZR, ZF, and medulla (in descending order, qualitatively). Analysis of adrenal lipid extracts by LC/MS did not detect 9-cis-retinoic acid (a potent RXR-ligand) but identified all-trans retinoic acid. Since C20 and C22 polyunsaturated fatty acids (PUFAs) can also activate RXR, subcellular availabilities of unesterified fatty acids were investigated by GC/MS. As results, arachidonic acid (C20:4), adrenic acid (C22:4), docosapentaenoic acid (C22:5), and cervonic acid (C22:6) were detected in the lipids extracted from each subcellular fraction. Thus, the RXR-agonizing PUFAs are available in all the main subcellular compartments considerably. The present findings not only shed light on the adrenal network of RXRs but also provide baseline information for further investigations of RXR heterodimers in the regulation of adrenal steroidogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Stroke is the consequence of limited blood flow to the brain with no established treatment to reduce the neurological deficits. Focusing on therapeutic protocols in targeting subventricular zone (SVZ) neurogenesis has been investigated recently. This study was designed to evaluate the effects of retinoic acid (RA)-pretreated Wharton's jelly mesenchymal stem cells (WJ-MSCs) in combination with triiodothyronine (T3) in the ischemia stroke model. Male Wistar rats were used to induce focal cerebral ischemia by middle cerebral artery occlusion (MCAO). There were seven groups of six animals: Sham, Ischemic, WJ-MSCs, RA-pretreated WJ-MSCs, T3, WJ-MSCs +T3, and RA-pretreated WJ-MSCs + T3. The treatment was performed at 24 h after ischemia, and animals were sacrificed one week later for assessments of retinoid X receptor β (RXRβ), brain-derived neurotrophic factor (BDNF), Sox2 and nestin in the SVZ. Pro-inflammatory cytokines in sera were measured at days four and seven after ischemia. RXRβ, BDNF, Sox2 and nestin had the significant expressions in gene and protein levels in the treatment groups, compared with the ischemic group, which were more vivid in the RA-pretreated WJ-MSCs + T3 (p ≤ 0.05). The same trend was also resulted for the levels of TNF-α and IL-6 at four days after ischemia (p ≤ 0.05). In conclusion, application of RA-pretreated WJ-MSCs + T3 could be beneficial in exerting better neurotrophic function probably via modulation of pro-inflammatory cytokines.

Brown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several "browning" pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Virus-induced exacerbations often lead to further impairment of lung function in chronic obstructive pulmonary disease. IL-15 is critical in antiviral immune responses. Retinoic acid (RA) signaling plays an important role in tissue maintenance and repair, particularly in the lung. We studied RA signaling and its relation to IL-15 in the lung during cigarette smoke (CS) exposure and influenza virus infection. In vivo studies show that RA signaling is diminished by long-term CS exposure or influenza virus infection alone, which is further attenuated during infection after CS exposure. RA receptor β (RARβ) is specifically decreased in the lung of IL-15 transgenic (overexpression; IL-15Tg) mice, and a greater reduction in RARβ is found in these mice compared with wild-type (WT) mice after infection. RARβ is increased in IL-15 knockout (IL-15KO) mice compared with WT mice after infection, and the additive effect of CS and virus on RARβ down-regulation is diminished in IL-15KO mice. IL-15 receptor α (IL-15Rα) is increased and RARβ is significantly decreased in lung interstitial macrophages from IL-15Tg mice compared with WT mice. In vitro studies show that IL-15 down-regulates RARβ in macrophages via IL-15Rα signaling during influenza virus infection. These studies suggest that RA signaling is significantly diminished in the lung by CS exposure and influenza virus infection. IL-15 specifically down-regulates RARβ expression, and RARβ may play a protective role in lung injury caused by CS exposure and viral infections.

Background: Sebaceous cell carcinoma of the eyelid is a malignant tumor. However, the pathoetiology of sebaceous cell carcinoma is not clear. Retinoic acid (RA) signaling is essential for skin epidermal differentiation including the eyelids. In this study, we investigate the expression of β-catenin, RA-binding proteins and RA receptors in sebaceous cell carcinoma of the eyelid and try to estimate their influence on its pathoetiology., Methods: Retrospective, noncomparative, consecutive interventional case series. Sixteen cases of eyelid sebaceous gland carcinoma who received tumor excision at our hospital between 2001 and 2011 were included. Immunohistochemical staining for β-catenin, cellular retinoic acid binding protein 1 (CRABP1), cellular retinoic acid binding protein 2 (CRABP2), fatty acid-binding protein 5 (FABP5), retinoic acid receptors (RAR-α, -β, -γ), and retinoid X receptors (RXR-α, -β, -γ) was performed on tissue samples obtained from tumor excision., Results: Of the 16 sebaceous cell carcinoma cases reviewed, six were male and 10 female. The mean follow-up period was 6.7 ± 3.66 years (range, 0.3-13 years). Of these 16 cases, the expression of β-catenin was significantly increased in sebaceous cell carcinoma cases. CRABP1 was similarly expressed in the sebaceous cell carcinoma and control groups. CRABP2 and FABP5 were expressed in hair follicles of lid skin in both groups, whereas the CRABP2 and FABP5 were aberrantly expressed in the tumor cells of the sebaceous glands. Notably, the expression of retinoic acid receptor (RAR-β) and retinoid X receptors (RXR-β, -γ) was significantly upregulated in sebaceous cell carcinoma of the eyelids., Conclusions: Our findings indicate that retinoic acid signaling is related to the pathogenesis of sebaceous cell carcinoma of the eyelids.

The prefrontal cortex shows structural and functional alterations in mood disorders. Retinoid signaling, brain-derived neurotrophic factor (BDNF), and its receptor TrkB are reported to be involved in depression. Here, we found that mRNA levels of key elements of retinoid signaling were significantly reduced in the postmortem dorsolateral prefrontal cortex/anterior cingulate cortex (ACC) from elderly depressed patients who did not die from suicide. Decreased mRNA levels of BDNF and TrkB isoforms were also found. Similar alterations were observed in rats subjected to chronic unpredictable mild stress. Along with neurons immunopositive for both retinoic acid receptor-α (RARα) and TrkB, a positive correlation between mRNA levels of the 2 receptors was found in the ACC of control subjects but not of depressed patients. In vitro studies showed that RARα was able to bind to and transactivate the TrkB promoter via a putative RA response element within the TrkB promoter. In conclusion, the retinoid and BDNF-TrkB signaling in the prefrontal cortex are compromised in mood disorders, and the transcriptional upregulation of TrkB by RARα provide a possible mechanism for their interaction. The retinoid signaling pathway that may activate TrkB expression will be an alternative novel target for BDNF-based antidepressant treatment., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

This exhaustive in silico study looks into the molecular interactions of phthalates and their metabolites with human peroxisome proliferator-activated receptor (hPPAR) and retinoid X receptor (hRXR) α, β and γ subtypes--the nuclear receptor proteins function as transcription factors by regulating the expression of downstream genes. Apart from the much discussed plasticizer bisphenol A, we examined the binding affinities of 15 common diphthalates and their monophthalates, natural (linoleic acid, conjugated linoleic acid) and synthetic (bezafibrate, pioglitazone, GW 50156) ligands with hPPARs. In addition to these phthalates, specific natural (retinoic and phytanic acids) and synthetic (bexarotene, rosiglitazone) ligands were examined with hRXRs. The Maestro, Schrödinger Suite 2012 was used for the molecular docking study. In general, natural ligands of hPPAR showed less binding efficiencies than phthalic acid esters and drugs. The diphthalate di-iso-decyl phthalate showed the highest G score (-9.99) with hPPAR (γ), while its monophthalate (mono-iso-decyl phthalate) showed a comparatively less G score (-9.56). Though the PPAR modulator GW 50156 showed strong affinity with all hPPAR subtypes, its highest G score (-12.43) was with hPPARβ. Hazardous di(2-ethylhexyl)phthalate generally showed a greater preference to hRXRs than hPPARs, but its highest G score (-10.87) was with hRXRα; while its monophthalate (Mono(2-ethylhexyl)phthalate) showed a lesser G score (-8.59). The drug bexarotene showed the highest G score (-13.32) with hRXRβ. Moreover, bisphenol A showed more affinity towards hRXR. Briefly, this study gives an overview on the preference of phthalic acid esters, natural and synthetic ligands on to hPPAR and hRXR subtypes, which would lead to further in vitro mechanistic as well as in vivo preclinical and clinical studies., (Copyright © 2013 John Wiley & Sons, Ltd.)

How Retinoid X receptors (RXR) and thyroid hormone receptors (TR) interact on negative TREs and whether RXR subtype specificity is determinant in such regulations is unknown. In a set of functional studies, we analyzed RXR subtype effects in T3-dependent repression of hypothalamic thyrotropin-releasing hormone (Trh). Two-hybrid screening of a hypothalamic paraventricular nucleus cDNA bank revealed specific, T3-dependent interaction of TRs with RXRβ. In vivo chromatin immuno-precipitation showed recruitment of RXRs to the TRE-site 4 region of the Trh promoter in the absence of T3. In vivo overexpression of RXRα in the mouse hypothalamus heightened T3-independent Trh transcription, whereas RXRβ overexpression abrogated this activity. Loss of function of RXRα and β by shRNAs induced inverse regulations. Thus, RXRα and RXRβ display specific roles in modulating T3-dependent regulation of Trh. These results provide insight into the actions of these different TR heterodimerization partners within the context of a negatively regulated gene., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Aberrant histone acetylation plays an essential role in the neoplastic process via the epigenetic silencing of tumour suppressor genes (TSGs); therefore, the inhibition of histone deacetylases (HDAC) has become a promising target in cancer therapeutics. To investigate the correlation of histone acetylation with clinicopathological features and TSG expression, we examined the expression of acetylated H3 (AcH3), RARβ2, E-cadherin, and β-catenin by immunohistochemistry in 65 cervical squamous cell carcinoma patients. The results revealed that the absence of AcH3 was directly associated with poor histological differentiation and nodal metastasis as well as reduced/negative expression of RARβ2, E-cadherin, and β-catenin in clinical tumour samples. We further demonstrated that the clinically available HDAC inhibitors valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), in combination with all-trans retinoic acid (ATRA), can overcome the epigenetic barriers to transcription of RARβ2 in human cervical cancer cells. Chromatin immunoprecipitation analysis showed that the combination treatment increased the enrichment of acetylated histone in the RARβ2-RARE promoter region. In view of these findings, we evaluated the antitumor effects induced by combined VPA and ATRA treatment in a xenograft model implanted with poorly differentiated human squamous cell carcinoma. Notably, VPA restored RARβ2 expression via epigenetic modulation. Additive antitumour effects were produced in tumour xenografts by combining VPA with ATRA treatment. Mechanistically, the combination treatment reactivated the expression of TSGs RARβ2, E-cadherin, P21 (CIP1) , and P53 and reduced the level of p-Stat3. Sequentially, upregulation of involucrin and loricrin, which indicate terminal differentiation, strongly contributed to tumour growth inhibition along with partial apoptosis. In conclusion, targeted therapy with HDAC inhibitors and RARβ2 agonists may represent a novel therapeutic approach for patients with cervical squamous cell carcinoma.

Background: A malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells., Methods: This study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells., Results: ACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC₅₀ values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21(CIP1), while decreasing the levels of cyclin D1., Conclusion: ACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Transplantation of antigenic-separated stem cells for human cardiovascular diseases such as myocardial infarction needs to be supported by experimental studies that allow refinement of the procedure. In this study we investigated optimising a protocol for the expansion and subsequent differentiation of human umbilical cord blood (HUCB) derived CD133(+) stem cells into a cardiomyocyte-like lineage. CD133(+) cells from HUCB were selected first by immunomagnetic separation and their purity was confirmed by flow cytometry analysis. For expansion and differentiation we developed a novel culture medium recipe that involves sequential signalling factors. Briefly, CD133(+) cells were expanded for 6 days under optimal serum-free conditions in combination with fibronectin and assessed by microscopy and AlamarBlue proliferation assay. Expanded CD133(+) cells were then plated in a cardiac differentiation promoting medium and cultured up to 4 weeks. With this protocol HUCB-CD133(+) cells can be regularly expanded in serum-free medium to obtain recovery and growth in vitro up to 6 folds. The addition of recombinant human thrombopoietin to the remaining factors of the expanding medium was associated with larger cell expansion. Expanded UCB CD133(+) cells showed a cardiomyocyte-like phenotype following differentiation in vitro through expressing intracellular cardiac specific markers including cardiac-specific α-actin, myosin heavy chain and troponin I. This change in phenotype was associated with the expression of cardiac-specific transcription factors Gata-4 and MEF2C. In addition, the change in phenotype was associated with an upregulation of nuclear receptor transcription factors including PPAR α, PPARγ, RXR α and RXRβ. We believe our protocol represents a significant advancement and overcome the technical hurdle of deriving cardiomyogenic-like cells from HUCB CD133(+) stem cells. In addition, it has the required attributes of simplicity and consistency. This will permit more robust manipulation of these cells towards better engraftment and repair in patients with myocardial infarction.

Long-chain acyl-CoA synthetases (ACSL) play key roles in fatty acid metabolism in liver and other metabolic tissues in an isozyme-specific manner. In this study, we examined the effects of a fructose-enriched diet on expressions of ACSL isoforms in the liver of hamsters. We showed that the fructose diet markedly reduced the mRNA and protein expressions of ACSL3 in hamster liver without significant effects on other ACSLs. The decrease in ACSL3 abundance was accompanied by a reduction in ACSL-catalyzed synthesis of arachidonyl-CoA and oleoyl-CoA in liver homogenates of hamsters fed the fructose diet as opposed to normal diet. We further showed that fructose diet specifically reduced expressions of three key components of the LXR signaling pathway, namely, liver X receptor (LXR)α, LXRβ, and retinoid X receptor (RXR)β. Exogenous expression and activation of LXRα/β increased hamster ACSL3 promoter activities in a LXR-responsive element (LXRE)-dependent fashion. Finally, we showed that treating hamsters with LXR agonist GW3965 increased hepatic ACSL3 expression without affecting other ACSL isoforms. Furthermore, the ligand-induced increases of ACSL3 expression were accompanied with the reduction of hepatic triglyceride levels in GW3965-treated hamster liver. Altogether, our studies demonstrate that fructose diet has a negative impact on LXR signaling pathway in liver tissue and reduction of ACSL3 expression/activity could be a causal factor for fructose-induced hepatic steatosis.

An endogenous time-keeping mechanism controls circadian biological rhythms in mammals. Previously, we showed that vitamin A deficiency modifies clock BMAL1 and PER1 as well as BDNF and neurogranin daily rhythmicity in the rat hippocampus when animals are maintained under 12-h-light:12-h-dark conditions. Retinoic acid nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been detected in the same brain area. Our objectives were (a) to analyze whether RARα, RARβ and RXRβ exhibit a circadian variation in the rat hippocampus and (b) to investigate the effect of a vitamin-A-deficient diet on the circadian expression of BMAL1, PER1 and retinoic acid receptors (RARs and RXRβ) genes. Holtzman male rats from control and vitamin-A-deficient groups were maintained under 12-h-light:12-h-dark or 12-h-dark:12-h-dark conditions during the last week of treatment. RARα, RARβ, RXRβ, BMAL1 and PER1 transcript and protein levels were determined in hippocampus samples isolated every 4 h in a 24-h period. Regulatory regions of RARs and RXRβ genes were scanned for clock-responsive sites, while BMAL1 and PER1 promoters were analyzed for retinoic acid responsive elements and retinoid X responsive elements. E-box and retinoid-related orphan receptor responsive element sites were found on regulatory regions of retinoid receptors genes, which display an endogenously controlled circadian expression in the rat hippocampus. Those temporal profiles were modified when animals were fed with a vitamin-A-deficient diet. Similarly, the nutritional vitamin A deficiency phase shifted BMAL1 and abolished PER1 circadian expression at both mRNA and protein levels. Our data suggest that vitamin A deficiency may affect the circadian expression in the hippocampus by modifying the rhythmic profiles of retinoic acid receptors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The circadian expression of clock and clock-controlled cognition-related genes in the hippocampus would be essential to achieve an optimal daily cognitive performance. There is some evidence that retinoid nuclear receptors (RARs and RXRs) can regulate circadian gene expression in different tissues. In this study, Holtzman male rats from control and vitamin A-deficient groups were sacrificed throughout a 24-h period and hippocampus samples were isolated every 4 or 5 h. RARα and RXRβ expression level was quantified and daily expression patterns of clock BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins, as well as temporal expression of cognition-related RC3 and BDNF genes were determined in the hippocampus of the two groups of rats. Our results show significant daily variations of BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins and, consequently, daily oscillating expression of RC3 and BDNF genes in the rat hippocampus. Vitamin A deficiency reduced RXRβ mRNA level as well as the amplitude of PER1, REVERB gene, and REVERB protein rhythms, and phase-shifted the daily peaks of BMAL1 and RORα mRNA, RORα protein, and RC3 and BDNF mRNA levels. Thus, nutritional factors, such as vitamin A and its derivatives the retinoids, might modulate daily patterns of BDNF and RC3 expression in the hippocampus, and they could be essential to maintain an optimal daily performance at molecular level in this learning-and-memory-related brain area., (Copyright © 2012 Wiley Periodicals, Inc.)

We previously identified transthyretin (TTR) and its posttranslational modifications as a down-regulated marker in mycosis fungoides (MF), a benign subtype of cutaneous T-cell lymphoma (CTCL). In order to more precisely understand the biological role of TTR in the etiology of MF, it is essential to clarify the pathways of progression by identifying further interacting proteins. This study is the first to combine blue native polyacrylamide gel electrophoresis (BN-PAGE) with surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to detect new TTR interaction partners and to determine whether these TTR interaction partners can themselves be used as biomarkers. By this procedure, apolipoprotein A1, which was additionally found to be down-regulated in the serum of MF patients, apolipoprotein A4, retinol binding protein 4 (RBP-4), and retinoid X receptor β (RXR-β) were identified as interaction partners of TTR. The RXR family plays a role in cell differentiation and proliferation and is known to be the target of bexarotene, which is used in the treatment of CTCL. In conclusion, the combination of BN-PAGE and SELDI-TOF-MS used in this study allowed for the detection of protein interaction partners, which, in the case of RBP-4 and RXR, indicated a connection between the common tumor marker TTR and tumor progression in CTCL.

Objective: To determine whether arsenic inhibits transcriptional activation of the liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers, thereby impairing cholesterol efflux from macrophages and potentially contributing to a proatherogenic phenotype., Methods and Results: Arsenic is an important environmental contaminant and has been linked to an increased incidence of atherosclerosis. Previous findings showed that arsenic inhibits transcriptional activation of type 2 nuclear receptors, known to heterodimerize with RXR. Environmentally relevant arsenic doses decrease the LXR/RXR ligand-induced expression of the LXR target genes (ABCA1 and SREBP-1c). Arsenic failed to decrease cAMP-induced ABCA1 expression, suggesting a selective LXR/RXR effect. This selectivity correlated with the ability of arsenic to decrease LXR/RXR ligand-induced, but not cAMP-induced, cholesterol efflux. By using chromatin immunoprecipitation assays, we found that arsenic inhibits the ability of LXR/RXR ligands to induce activation markers on the ABCA1 and SREBP-1c promoters and blocks ligand-induced release of the nuclear receptor coexpressor (NCoR) from the promoter. Arsenic did not alter the ability of LXR to transrepress inflammatory gene transcription, further supporting our hypothesis that RXR is the target for arsenic inhibition., Conclusions: Exposure to arsenic enhances the risk of atherosclerosis. We present data that arsenic inhibits the transcriptional activity of the liver X receptor, resulting in decreased cholesterol-induced gene expression and efflux from macrophages. Therefore, arsenic may promote an athersclerotic environment by decreasing the ability of macrophages to efflux excess cholesterol, thereby favoring increased plaque formation.

Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all-trans-retinoic acid (ATRA) pre-induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01-100 micromol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 micromol/L ATRA pre-induction significantly improved neuronal differentiation efficiency and neural-cell survival. Compared with MNM alone induced neural-like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule-associated protein-2 (MAP-2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line-derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre-induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRalpha and RXRgamma (and to a lesser extent, RXRbeta) were weakly expressed in MSCs. But the expression of RARalpha and RARgamma was readily detectable, whereas RARbeta was undetectable. However, at 24 h after ATRA treatment, the expression of RARbeta, not RARalpha or RARgamma, increased significantly. We further found the subnuclear redistribution of RARbeta in differentiated neurons, suggesting that RARbeta may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre-activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.

Mechanisms regulating CYP4F genes remain under investigation, although characterization of CYP4F regulatory modalities would facilitate the discovery of new drug targets. This present study shows that all-trans- and 9-cis-retinoic acids can inhibit CYP4F11 expression in human keratinocyte-derived HaCaT cells. Transrepression of many genes by retinoic acids is mediated by interactions between retinoid receptors and the activator protein 1 (AP-1) complex. Proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta, which can activate the AP-1 complex, induce CYP4F11 transcription in HaCaT cells. The c-Jun N-terminal kinase (JNK)-specific inhibitor 1,9-pyrazoloanthrone (SP600125) blocked the induction of CYP4F11 by both cytokines, indicating involvement of the JNK pathway. Furthermore, TNF-alpha failed to induce CYP4F11 transcription when HaCaT cells were preincubated with retinoic acids. Retinoic acids are ligands for the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RXR agonist 6-(1(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl) nicotinic acid (LG268) greatly induced CYP4F11 transcription, whereas the RAR agonist 4-(2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB) markedly inhibited CYP4F11 transcription, indicating that down-regulation of CYP4F11 transcription by retinoic acid is mediated by RARs and may also be related to ligand competition for RXRs. Thus, the CYP4F11 gene is positively regulated by multiple signaling pathways in HaCaT keratinocytes, including RXR and JNK signaling pathways.

Down-regulation of transcription of the MHC class I genes in HPV16 tumorigenic cells is partly due to HPV16E7 associated with the MHC class I promoter and repressed chromatin activation. In this study, we further demonstrated that HPV16E7 is physically associated with a putative RXRbeta binding motif (GGTCA) of the proximal promoter of the MHC class I genes by using reporter transcriptional assays and chromatin immunoprecipitation assays. Our data also provide evidence that HPV16E7 inhibits TNF-alpha-induced up-regulation of MHC class I transcription by impaired nuclear translocation of NF-kappaB. More importantly, CaSki tumor cells treated with TSA and transfected with the constitutively active mutant form of IKK-alpha (which can activate NF-kappaB directly) showed a maximal level of up-regulation of MHC-I expression. Taken together, our results suggest that HPV16E7 may employ two independent mechanisms to ensure that either the constitutive or inducible transcription of MHC class I genes is down-regulated.

Animals can adapt their behavior to predictable temporal fluctuations in the environment through both, memory-and-learning processes and an endogenous time-keeping mechanism. Hippocampus plays a key role in memory and learning and is especially susceptible to oxidative stress. In compensation, antioxidant enzymes activity, such as Catalase (CAT) and Glutathione peroxidase (GPx), has been detected in this brain region. Daily rhythms of antioxidant enzymes activity, as well as of glutathione and lipid peroxides levels, have been described in brain. Here, we investigate day/night variations in lipoperoxidation, CAT, and GPx expression and activity, as well as the temporal fluctuations of two key components of the endogenous clock, BMAL1 and PER1, in the rat hippocampus and evaluate to which extent vitamin A deficiency may affect their amplitude or phase. Holtzman male rats from control, vitamin A-deficient, and vitamin A-refed groups were sacrificed throughout a 24-h period. Daily levels of clock proteins, lipoperoxidation, CAT and GPx mRNA, protein, and activity, were determined in the rat hippocampus obtained every 4 or 5 h. Gene expression of RARalpha and RXRbeta was also quantified in the hippocampus of the three groups of rats. Our results show significant daily variations of BMAL1 and PER1 protein expression. Rhythmic lipoperoxidation, CAT, and GPx, expression and activity, were also observed in the rat hippocampus. Vitamin A deficiency reduced RXRbeta mRNA level, as well as the amplitude of BMAL1 and PER1 daily oscillation, phase-shifted the daily peak of lipoperoxidation, and had a differential effect on the oscillating CAT and GPx mRNA, protein, and activity. Learning how vitamin A deficiency affects the circadian gene expression in the hippocampus may have an impact on the neurobiology, nutritional and chronobiology fields, emphasizing for the first time the importance of nutritional factors, such as dietary micronutrients, in the regulation of circadian parameters in this brain memory-and-learning-related region., ((c) 2009 Wiley-Liss, Inc.)

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.

Somatic, targeted inactivation of the retinoid X receptor beta gene (Rxrb) in Sertoli cells (SC; yielding Rxrb(Ser-/-) mutants) leads to failure of spermatid release, accumulation of cholesterol esters and, subsequently, testis degeneration. These abnormalities are identical, in their nature and kinetics, to those observed upon inactivating Rxrb in the whole organism, thereby demonstrating that all reproductive functions of RXRB are carried out in SC. The Rxrb(Ser-/-) testis degeneration is a consequence of a cholesterol ester cell overload occurring in SC in response to reduced ABCA1- and SCARB1-mediated cholesterol efflux. The failure of spermiation was also reported in mice lacking the retinoic acid (RA) receptor-alpha (RARA) in SC (Rara(Ser-/-) mutants) and represents, in addition, a feature of vitamin A deficiency that can be readily induced in mice lacking the lecithin:retinol acyltransferase (Lrat(-/-) mutants). Altogether, these findings support the conclusion that RXRB heterodimerized with a RA-liganded RARA transduces signals required in SC for spermatid release.

Obesity is associated with impaired fatty acid (FA) oxidation and increased de novo hepatic lipogenesis that may contribute to the development of hypertriglyceridemia, an important risk factor for the development of cardiovascular disease. Strategies to improve hepatocyte FA metabolism, including dietary interventions, are therefore important for the prevention of obesity-associated co-morbidities. Farnesol is consumed in the diet as a component of plant products. In the present study, we administered farnesol orally to rats for seven days and found significantly reduced serum triglyceride concentrations compared with controls. Potential mechanisms underlying the hypotriglyceridemic effect of farnesol were investigated using clone-9 cultured rat hepatocytes. Farnesol significantly upregulated expression of peroxisome proliferator-activated receptor alpha (PPARalpha) and the PPARalpha-regulated genes fatty acyl-CoA oxidase and carnitine palmitoyl transferase 1a, suggesting that increased hepatic FA oxidation may contribute to serum triglyceride lowering in rats. Farnesol did not change SREBP-1c mRNA levels, but significantly down-regulated fatty acid synthase (FAS) mRNA and protein levels and activity, indicating that attenuated lipogenesis may also contribute to hypotriglyceridemic effects of farnesol in vivo. Rescue experiments revealed that down-regulation of FAS by farnesol was not related to activation of PPARalpha, but rather was caused by a 9-cis retinoic acid mediated mechanism that involved down-regulation of retinoid X receptor beta. Diets rich in plant products are associated with a lower risk of cardiovascular disease. Our findings suggest that farnesol may contribute to this protective effect by lowering serum TG levels.

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands are important regulators of lipid metabolism, inflammation, and diabetes. We previously demonstrated that anucleate human platelets express the transcription factor PPARgamma and that PPARgamma ligands blunt platelet activation. To further understand the nature of PPARgamma in platelets, we determined the platelet PPARgamma isoform(s) and investigated the fate of PPARgamma following platelet activation. Our studies demonstrated that human platelets contain only the PPARgamma1 isoform and after activation with thrombin, TRAP, ADP or collagen PPARgamma is released from internal stores. PPARgamma release was blocked by a cytoskeleton inhibitor, Latrunculin A. Platelet-released PPARgamma was complexed with the retinoid X receptor (RXR) and retained its ability to bind DNA. Interestingly, the released PPARgamma and RXR were microparticle associated and the released PPARgamma/RXR complex retained DNA-binding ability. Additionally, a monocytic cell line, THP-1, is capable of internalizing PMPs. Further investigation following treatment of these cells with the PPARgamma agonist, rosiglitazone and PMPs revealed a possible transcellular mechanism to attenuate THP-1 activation. These new findings are the first to demonstrate transcription factor release from platelets, revealing the complex spectrum of proteins expressed and expelled from platelets, and suggests that platelet PPARgamma has an undiscovered role in human biology.

Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) gamma, PPARbeta, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRalpha and RXRbeta. RXR ligands inhibit platelet aggregation and TXA(2) release to ADP and the TXA(2) receptors, but only weakly to collagen. ADP and TXA(2) both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families.

Because RXR plays a significant role in nuclear receptor signaling as a common heterodimeric partner for TR, PPAR, RAR, VDR, LXR and others, the ability of RXRbeta ligand binding domain (LBD) to interact with coregulator peptides bearing LXXLL or other interaction motifs was investigated using time-resolved fluorescence resonance energy transfer (TR-FRET). The random phage display peptide D22 and peptides derived from PGC1alpha, SRC1-4, SRC2-3, PRIP/RAP250 and RIP140 yielded the highest TR-FRET signal with RXRbeta LBD in the presence of saturating 9-cis retinoic acid (9-cisRA). Several peptides including D22, PGC1alpha, SRC3-2, PRIP/RAP250 and SRC1-4 also formed a complex with RXRbeta LBD in the presence of all-trans retinoic acid (at-RA) and the fatty acids, phytanic acid (PA) and docosahexaenoic acid (DHA). Determination of the dose dependency (EC50) of these compounds to recruit D22 to RXRbeta LBD indicated that the rank order potency was 9-cisRA>PA>at-RA>DHA. The ligands 9-cisRA and at-RA yielded an overall higher fold-change in D22 recruitment to RXRbeta LBD suggesting that more RXRbeta LBD-D22 complex was formed in the presence of these ligands under the assay conditions tested. The statistical parameter Z' factor for 9-cisRA-induced recruitment of D22 to RXRbeta LBD was 0.6 after 2h incubation, indicating a robust methodology that could be applied to high throughput screening. These results demonstrate that RXRbeta occupied with the fatty acid ligands, DHA and PA, can recruit coactivator peptides in a ligand-dependent manner.

Sertoli cells (SC) are instrumental to stem spermatogonia differentiation, a process that critically depends on retinoic acid (RA). We show here that selective ablation of RA receptor alpha (RARalpha) gene in mouse SC, singly (Rara(Ser-/-) mutation) or in combination with RARbeta and RARgamma genes (Rara/b/g(Ser-/-) mutation), abolishes cyclical gene expression in these cells. It additionally induces testis degeneration and delays spermatogonial expression of Stra8, two hallmarks of RA deficiency. As identical defects are generated upon inactivation of RARalpha in the whole organism, our data demonstrate that all the functions exerted by RARalpha in male reproduction are Sertoli cell-autonomous. They further indicate that RARalpha is a master regulator of the cyclical activity of SC and controls paracrine pathways required for spermatogonia differentiation and germ cell survival. Most importantly, we show that the ablation of all RXR (alpha, beta and gamma isotypes) in SC does not recapitulate the phenotype generated upon ablation of all three RARs, thereby providing the first evidence that RARs exert functions in vivo independently of RXRs.

We have demonstrated that cytokine thymic stromal lymphopoietin (TSLP), whose expression is rapidly induced upon keratinocyte-selective ablation of retinoid X receptors (RXRs) -alpha and -beta in the mouse (RXRalphabeta(ep-/-) mice), plays a key role in initiating a skin and systemic atopic dermatitis-like phenotype. We show here that topical application of the physiologically active ligand [1alpha,25-(OH)(2)D(3); calcitriol] of the vitamin D receptor, or of its low-calcemic analog MC903 (calcipotriol; Dovonex), induces TSLP expression in epidermal keratinocytes, which results in an atopic dermatitis-like syndrome mimicking that seen in RXRalphabeta(ep-/-) mutants and transgenic mice overexpressing TSLP in keratinocytes. Furthermore, topical application of retinoic acid receptor RARgamma-selective agonist BMS961 also induces TSLP expression either on its own or synergistically with 1alpha,25-(OH)(2)D(3). Our data demonstrate that RXR/vitamin D receptor and RXR/retinoic acid receptor-gamma heterodimers and their ligands cell-autonomously control the expression of TSLP in epidermal keratinocytes of the mouse. We propose molecular mechanisms through which vitamin D3 and retinoic acid signalings could be involved in the pathogenesis of atopic diseases.

Retinoic acid (RA) plays an important role in bone metabolism in vivo through osteoclast activation and bone resorption. Retinoid X-activated receptor beta (RXRbeta) has been implicated in the genetic spinal defect of ossification of the posterior longitudinal ligament (OPLL). In this study, we examined the effects of 9-cis RA and all-trans RA (ATRA) on the proliferation, differentiation, and RXRbeta expression of the murine preosteoblastic cell line MC3T3-E1. Both 9-cis RA and ATRA dose-dependently inhibited the increase in total soluble protein content at concentrations of 10 and 100 nM after 4 and 8 d co-culture with MC3T3-E1 cells. The inhibitory effect of 9-cis RA was slightly stronger than that of ATRA. Histone H4 mRNA expression was dose-dependently suppressed by both RAs on day 1. Alkaline phosphatase activity was increased by both RAs at 10 and 100 nM concentrations on day 4, with 9-cis RA-induced activity slightly stronger than that of ATRA. Osteopontin mRNA expression was increased by both RAs on day 1, but was suppressed on day 4. Bone Gla protein mRNA expression was inhibited by 10 and 100 nM 9-cis RA and by 100 nM ATRA on day 14. RXRbeta mRNA expression was increased by 9-cis RA, an RXRbeta ligand, in a dose-dependent manner. Our results suggested that while both RAs suppressed proliferation and stimulated the maturation of preosteoblastic MC3T3-E1 cells, 9-cis RA was slightly more potent than ATRA. It also appeared that RAs may contribute to the development of heterotopic ossification, including OPLL.

Dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein (DARPP-32) is a key molecule for dopamine neurotransmission. The molecular mechanisms underlying the regulation of DARPP-32 in the developing brain remains elusive. Previous studies have shown that retinoids are capable of inducing DARPP-32 in striatal cell culture, suggesting that retinoids are candidate molecules for controlling DARPP-32 expression. In the present study, we first studied the expression profiles of retinoid receptors and their associated co-factors in the developing rat telencephalon by RT-PCR. The results showed that among the retinoid receptors, RARbeta and RXRgamma were nearly selectively expressed in the developing striatum. By contrast, the retinoid receptors associated transcriptional co-factors, including the co-repressors of N-CoR and SMRT, and the co-activators of SRC-1 and P/CAF, were ubiquitously expressed in the developing telencephalon. In light of the previous findings that DARPP-32 was inducible by retinoids in striatal culture, but not in cortical culture, we hypothesized that the striatum-selective RARbeta and RXRgamma may mediate DARPP-32 induction by retinoids. To test this hypothesis, we used the gain-of-function approach to ectopically express RARbeta and RXRgamma in the developing cerebral cortex that lacked these two retinoid receptors. Ectopic expression of RARbeta1, but not RXRgamma1, up-regulated DARPP-32 in the cortical explant culture. Notably, DARPP-32 was up-regulated only by the RARbeta1 isoform, but not by other RARbeta isoforms. Our study suggests that RARbeta signaling may regulate DARPP-32 gene expression by an isoform-specific mechanism in developing telencephalic neurons. The molecular diversity of RARbeta isoforms may underlie parts of the complex gene regulation by retinoids during neural development.

The retroviral v-ErbA oncoprotein is a highly mutated variant of the thyroid hormone receptor alpha (TRalpha), which is unable to bind T(3) and interferes with the action of TRalpha in mammalian and avian cancer cells. v-ErbA dominant-negative activity is attributed to competition with TRalpha for T(3)-responsive DNA elements and/or auxiliary factors involved in the transcriptional regulation of T(3)-responsive genes. However, competition models do not address the altered subcellular localization of v-ErbA and its possible implications in oncogenesis. Here, we report that v-ErbA dimerizes with TRalpha and the retinoid X receptor and sequesters a significant fraction of the two nuclear receptors in the cytoplasm. Recruitment of TRalpha to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A. These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.

Retinoid X receptors are frequently implicated in their role in embryonic development. However, there is no report regarding their expression in embryos with different qualities. In this study, expression pattern and levels of retinoid X receptor transcripts in different qualities and stages of in vitro-produced pre-implantation bovine embryos were examined using real-time polymerase chain reaction (PCR) techniques. Four independent cDNA pools per sample were prepared from mRNAs isolated from different developmental-stage embryos, after classifying them to be of good and bad quality. Primers were also designed to amplify products with PCR. Based on analysis, except retinoid X receptor gamma (RXRG) transcripts, the remaining two types (alpha and beta forms) were expressed in all samples with significantly higher (p < or = 0.05) levels until the eight-cell stage compared with the stages thereafter. However, the transcript levels for bad-quality embryos were consistently lower in all cases in comparison with good-quality embryos. RXRG was identified in 39% of the samples, but all in the pre-embryonic genome activation development stage. Therefore, strong expression patterns of these transcripts in earlier stages indicates their possible role during the maternal phase of embryo development, and the variations of copy number in embryos with different qualities substantiates their possible candidacy as potential quality markers. Moreover, identifying the transcript variations in different qualities and expression of RXRG at these stages is a novel input to the pre-implantation-stage knowledge.

The prohormone convertases (PCs) PC1 and PC2 are involved in the tissue-specific endoproteolytic processing of neuropeptide precursors within the secretory pathway. We previously showed that changes in thyroid status altered pituitary PC2 mRNA and that this regulation was due to triiodothyronine-dependent interaction of the thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large proximal region of the human PC2 promoter. In the current study, we examined the in vivo regulation of brain PC2 mRNA by thyroid status and found that 6-n-propyl-2-thiouracil-induced hypothyroidism stimulated, whereas thyroxine-induced hyperthyroidism suppressed, PC2 mRNA levels in the rat hypothalamus and cerebral cortex. To address the mechanism of T3 regulation of the PC2 gene, we used human PC2 (hPC2) promoter constructs transiently transfected into GH3 cells and found that triiodothyronine negatively and 9-cis-retinoic acid positively regulated hPC2 promoter activity. EMSAs, using purified TRalpha1 and retinoid X receptor-beta (RXRbeta) proteins demonstrated that TRalpha bound the distal putative nTRE-containing oligonucleotide in the PC2 promoter, and RXR bound to both nTRE-containing oligonucleotides. EMSAs with oligonucleotides containing deletion mutations of the nTREs demonstrated that the binding to TR and RXR separately is reduced, but specific binding to TR and RXR together persists even with deletion of each putative nTRE. We conclude that there are two novel TRE-like sequences in the hPC2 promoter and that these regions act in concert in a unique manner to facilitate the effects of thyroid hormone and 9-cis-retinoic acid on PC2.

Neuronal differentiation is a complex process involving the sequential expression of several factors. The important role of lipid molecules in brain development is well known. Many fatty acid cell signaling activities are mediated by peroxisome proliferator-activated receptors (PPARs). PPARs are ligand-activated transcription factors belonging to the steroid, thyroid and retinoid nuclear receptor superfamily. They are activated by fatty acids and their derivatives. Different isotypes of PPARs (alpha, beta/delta and gamma) have distinct physiological functions depending on their different ligand activation profiles and tissue distribution. PPARs have been involved in neural cell differentiation and death as well as in inflammation and neurodegeneration. Although PPARs have been described in neurons by in situ studies, the presence and possible modulation of these receptors during neuronal differentiation has not been explored yet. In this study we analyzed the expression of PPARs and of their heterodimeric partners, RXRs, in embryonic rat cortical neurons during their in vitro maturation. Our results demonstrate the presence of PPARs alpha, beta/delta and gamma and of RXRs beta and gamma. PPARalpha, beta/delta and gamma are differentially modulated during culture time suggesting that they may be involved in neuronal maturation. In particular, we point toward the PPARbeta/delta isotype as a key factor in neuronal differentiation.

The Col11a2 gene codes for alpha2(XI), a subunit of type XI collagen that is a critical component of the cartilage extracellular matrix. The 5' regulatory region of Col11a2 was subjected to deletional analysis to detect any regulatory element in addition to the two known chondrocyte-specific enhancer elements B/C and D/E. Deletion of the region from -342 to -242 bp reduced transcriptional activity to less than 50% of wild-type, but the sequence showed no independent ability to increase transcription from a minimal promoter. When cloned downstream of the D/E enhancer, however, a subsection of the sequence nearly doubled transcriptional activity and produced an additional 3-fold activation in response to RA (retinoic acid). A 6-bp direct repeat, separated by 4 bp (a DR-4 element) near the 5'-end of this region, was found to be essential for its activity, and was further shown to bind the RA X receptor beta in electrophoretic mobility-shift assays. The present study has revealed a novel RA-response element in Col11a2 that does not interact directly with the promoter, but instead requires the D/E enhancer to mediate transcriptional activation. Proteins bound at the enhancer, therefore, would be expected to affect the transcriptional response to RA. Such a system of regulation, particularly if found to be operating in other cartilage genes, could explain the conflicting responses RA produces in chondrocytes under different experimental conditions.

We show that mice expressing retinoid X receptor beta (RXRbeta) impaired in its transcriptional activation function AF-2 (Rxrb(af20) mutation) do not display the spermatid release defects observed in RXRbeta-null mutants, indicating that the role of RXRbeta in spermatid release is ligand-independent. In contrast, like RXRbeta-null mutants, Rxrb(af20) mice accumulate cholesteryl esters in Sertoli cells (SCs) due to reduced ABCA1 transporter-mediated cholesterol efflux. We provide genetic and molecular evidence that cholesterol homeostasis in SCs does not require PPARalpha and beta, but depends upon the TIF2 coactivator and RXRbeta/LXRbeta heterodimers, in which RXRbeta AF-2 is transcriptionally active. Our results also indicate that RXRbeta may be activated by a ligand distinct from 9-cis retinoic acid.