Retinoic Acid Receptor Binding Research Articles

Live cell single molecule imaging reveals concentration dependent changes to dynamic behavior of nuclear receptors RAR and RXR.

Type 2 Nuclear Receptors (T2NRs) like Retinoic Acid Receptor (RAR) are believed to require heterodimerization with a common factor, the Retinoid X Receptor (RXR), to bind chromatin and regulate its target genes. Here, we deploy stroboscopic photo-activation SPT (spaSPT) to understand the molecular dynamics of the RAR/RXR system and infer different diffusion states acquired by RAR and RXR in the nucleoplasm. These states occupied by RAR and RXR presumably exist in dynamic equilibrium. Next, we ask in a complex heterodimer driven transcriptional regulatory system such as this, how is the dynamic equilibrium shifted when either of the heterodimeric partner is in excess. We find that in live cells, increasing the number of RAR molecules and not RXR molecules leads to altered chromatin binding of RAR. While increasing the number of molecules of RAR and RXR both alter the chromatin binding of RXR. Since pharmacological modulation of the RAR/RXR system is an important avenue for cancer treatments, this study constitutes a step toward understanding how perturbations of this system affect chromatin binding and possibly downstream transcriptional regulatory function.

Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Transmural pressure signals through retinoic acid to regulate lung branching.

During development, the mammalian lung undergoes several rounds of branching, the rate of which is tuned by the relative pressure of the fluid within the lumen of the lung. We carried out bioinformatics analysis of RNA-sequencing of embryonic mouse lungs cultured under physiologic or sub-physiologic transmural pressure and identified transcription factor-binding motifs near genes whose expression changes in response to pressure. Surprisingly, we found retinoic acid (RA) receptor binding sites significantly overrepresented in the promoters and enhancers of pressure-responsive genes. Consistently, increasing transmural pressure activates RA signaling, and pharmacologically inhibiting RA signaling decreases airway epithelial branching and smooth muscle wrapping. We found that pressure activates RA signaling through the mechanosensor Yap. A computational model predicts that mechanical signaling through Yap and RA affects lung branching by altering the balance between epithelial proliferation and smooth muscle wrapping, which we test experimentally. Our results reveal that transmural pressure signals through RA to balance the relative rates of epithelial growth and smooth muscle differentiation in the developing mouse lung and identify RA as a previously unreported component in the mechanotransduction machinery of embryonic tissues.

Alpha-fetoprotein accelerates the progression of hepatocellular carcinoma by promoting Bcl-2 gene expression through an RA-RAR signalling pathway.

Previous studies have found that alpha‐fetoprotein (AFP) can promote the proliferation of hepatoma cells and accelerate the progression of hepatocellular carcinoma (HCC). However, the exact mechanism of action remains unclear. Recent bioinformatics studies have predicted the possible interaction between AFP and retinoic acid receptors (RARs). Thus, the purpose of this study was to investigate the molecular mechanism through which AFP promotes tumour cell proliferation by interfering with the RA‐RAR signal pathway. Our data indicated that AFP could significantly promote the proliferation and weaken ATRA‐induced apoptosis of hepatoma cells. Besides, cytoplasmic AFP interacts with RAR, disrupting its entrance into the nucleus, which in turn affects the expression of the Bcl‐2 gene. In addition, knockdown of AFP in HepG2 cells was synchronously associated with an incremental increase of RAR binding to DNA, as well as down‐regulation of Bcl‐2; the opposite effect was observed in AFP gene‐transfected HLE cells. Moreover, a similar effect of AFP was detected in tumour tissues with high serum AFP, but not in adjacent non‐cancerous liver tissues, or HCC tissues with low serum AFP levels. These results indicate that AFP acts as signalling molecule and prevents RAR from entering into the nucleus by interacting with RAR, thereby promoting the expression of Bcl‐2. Our data reveal a novel mechanism through which AFP regulates Bcl‐2 expression and further suggest that AFP may be used as a novel target for treating HCC.

Impact of Sustained Transforming Growth Factor-β Receptor Inhibition on Chromatin Accessibility and Gene Expression in Cultured Human Endometrial MSC.

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-β receptor (TGFβ-R) inhibitor, promotes expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFβ-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFβ-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RARβ). Selective RARβ inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture.

DNA recognition by retinoic acid nuclear receptors.

Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) to regulate gene expression. The heterodimer recognizes the genome via a large and diverse repertoire of DNA response elements. Assessing the binding mode of RAR and RXR with various DNA response elements is important for understanding how they select their binding site and how DNA sequence and topology allosterically regulate RAR function. A number of complementary assays are often employed for analysis of the binding mode. To biochemically and structurally characterize RAR and RXR-DNA complexes, we describe how to express and purify RAR and RXR-DNA binding domains (DBDs) and multidomain constructs. We also describe the use of electrospray ionization mass spectrometry (ESI MS) and isothermal titration calorimetry (ITC) that give information about stoichiometry and binding affinity, as well as our approaches for co-crystallization of RAR and RXR DBDs with DNA.

Risk variants disrupting enhancers of TH1 and TREG cells in type 1 diabetes

Genome-wide association studies (GWASs) have revealed 59 genomic loci associated with type 1 diabetes (T1D). Functional interpretation of the SNPs located in the noncoding region of these loci remains challenging. We perform epigenomic profiling of two enhancer marks, H3K4me1 and H3K27ac, using primary TH1 and TREG cells isolated from healthy and T1D subjects. We uncover a large number of deregulated enhancers and altered transcriptional circuitries in both cell types of T1D patients. We identify four SNPs (rs10772119, rs10772120, rs3176792, rs883868) in linkage disequilibrium (LD) with T1D-associated GWAS lead SNPs that alter enhancer activity and expression of immune genes. Among them, rs10772119 and rs883868 disrupt the binding of retinoic acid receptor α (RARA) and Yin and Yang 1 (YY1), respectively. Loss of binding by YY1 also results in the loss of long-range enhancer-promoter interaction. These findings provide insights into how noncoding variants affect the transcriptomes of two T-cell subtypes that play critical roles in T1D pathogenesis.

The Potential Mechanism of Bufadienolide-Like Chemicals on Breast Cancer via Bioinformatics Analysis.

Bufadienolide-like chemicals are mostly composed of the active ingredient of Chansu and they have anti-inflammatory, tumor-suppressing, and anti-pain activities; however, their mechanism is unclear. This work used bioinformatics analysis to study this mechanism via gene expression profiles of bufadienolide-like chemicals: (1) Differentially expressed gene identification combined with gene set variation analysis, (2) similar small -molecule detection, (3) tissue-specific co-expression network construction, (4) differentially regulated sub-networks related to breast cancer phenome, (5) differentially regulated sub-networks with potential cardiotoxicity, and (6) hub gene selection and their relation to survival probability. The results indicated that bufadienolide-like chemicals usually had the same target as valproic acid and estradiol, etc. They could disturb the pathways in RNA splicing, the apoptotic process, cell migration, extracellular matrix organization, adherens junction organization, synaptic transmission, Wnt signaling, AK-STAT signaling, BMP signaling pathway, and protein folding. We also investigated the potential cardiotoxicity and found a dysregulated subnetwork related to membrane depolarization during action potential, retinoic acid receptor binding, GABA receptor binding, positive regulation of nuclear division, negative regulation of viral genome replication, and negative regulation of the viral life cycle. These may play important roles in the cardiotoxicity of bufadienolide-like chemicals. The results may highlight the potential anticancer mechanism and cardiotoxicity of Chansu, and could also explain the ability of bufadienolide-like chemicals to be used as hormones and anticancer and vasoprotectives agents.

A novel retinoid X receptor agonist, UAB30, inhibits rhabdomyosarcoma cells in vitro

BackgroundWhile patients with early-stage rhabdomyosarcoma (RMS) have seen steady improvement in prognosis over the last 50 y, those with advanced-stage or high-grade disease continue to have a dismal prognosis. Retinoids have been shown to cause growth suppression and terminal differentiation in RMS cells, but the toxicities associated with retinoic acid limit its use. Rexinoids provide an alternative treatment approach to retinoic acid. Rexinoids primarily bind the retinoid X receptor with minimal retinoic acid receptor binding, the entity responsible for many of the toxicities of retinoid therapies. UAB30 is a novel rexinoid with limited toxicities. We hypothesized that UAB30 would lead to decreased cell survival in RMS. Materials and methodsTwo RMS cell lines, one embryonal (RD) subtype and one alveolar (St. Jude Cancer Research Hospital 30) subtype, were used. Cells were treated with UAB30, and cytotoxicity, proliferation, mobility, and apoptosis were evaluated. ResultsUAB30 significantly decreased RMS tumor cell viability and proliferation. Invasion, migration, and attachment-independent growth were reduced following UAB30 treatment. UAB30 also resulted in apoptosis and G1 cell cycle arrest. UAB30 affected both the alveolar and embryonal RMS cell lines in a similar fashion. ConclusionsThe results of these studies suggest a potential therapeutic role for the low-toxicity synthetic retinoid X receptor selective agonist, UAB30, in RMS treatment.

Detection of genomic structural variations in Guizhou indigenous pigs and the comparison with other breeds.

Genomic structural variation (SV) is noticed for the contribution to genetic diversity and phenotypic changes. Guizhou indigenous pig (GZP) has been raised for hundreds of years with many special characteristics. The present paper aimed to uncover the influence of SV on gene polymorphism and the genetic mechanisms of phenotypic traits for GZP. Eighteen GZPs were chosen for resequencing by Illumina sequencing platform. The confident SVs of GZP were called out by both programs of pindel and softSV simultaneously and compared with the SVs deduced from the genomic data of European pig (EUP) and the native pig outside of Guizhou, China (NPOG). A total of 39,166 SVs were detected and covered 27.37 Mb of pig genome. All of 76 SVs were confirmed in GZP pig population by PCR method. The SVs numbers in NPOG and GZP were about 1.8 to 1.9 times higher than that in EUP. And a SV hotspot was found out from the 20 Mb of chromosome X of GZP, which harbored 29 genes and focused on histone modification. More than half of SVs was positioned in the intergenic regions and about one third of SVs in the introns of genes. And we found that SVs tended to locate in genes produced multi-transcripts, in which a positive correlation was found out between the numbers of SV and the gene transcripts. It illustrated that the primary mode of SVs might function on the regulation of gene expression or the transcripts splicing process. A total of 1,628 protein-coding genes were disturbed by 1,956 SVs specific in GZP, in which 93 GZP-specific SV-related genes would lose their functions due to the SV interference and gathered in reproduction ability. Interestingly, the 1,628 protein-coding genes were mainly enriched in estrogen receptor binding, steroid hormone receptor binding, retinoic acid receptor binding, oxytocin signaling pathway, mTOR signaling pathway, axon guidance and cholinergic synapse pathways. It suggested that SV might be a reason for the strong adaptability and low fecundity of GZP, and 51 candidate genes would be useful for the configuration phenotype in Xiang pig breed.

ヒト顎下腺由来腺癌細胞株(HSG) レチノイン酸レセプターと レチノイン酸レスポンスエレメント における との結合

ヒト顎下腺由来腺癌細胞株(HSG) レチノイン酸レセプターと レチノイン酸レスポンスエレメント における との結合

A Therapeutic TCR Mimic Monoclonal Antibody for Intracellular PRAME Protein in Leukemias

Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-κB Response Elements.

ABSTRACTTriggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements [RARE], cyclic AMP [cAMP] response elements [CRE], NF-κB binding sites [kB], serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets the preinduction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In predifferentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation.IMPORTANCE Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of primitive neural stem cells carrying resting cytomegalovirus genomes, we show that costimulation of protein kinase A and C-delta signaling cascades in conjunction with retinoic acid-induced neuronal differentiation brings about progeny virus propagation. Iterated DNA binding sites for retinoic acid receptor, CREB, and NF-κB family members in the cytomegalovirus major enhancer are at the crux in the pathway to HCMV activation. The stimulated CREB and NF-κB binding site combination vigorously initiates and amplifies the active cytomegalovirus infection and cooperates with activated retinoic acid receptor binding sites to further promote viral proliferation and spread between differentiated cells. These results support a paradigm in which a specific combination of stimuli coupled with cellular differentiation satisfies a core cis-activating code that unlocks enhancer silence to repower the cycle of cytomegalovirus propagation.

Alterations in Retinoic Acid Receptors in Non-Small Cell Lung Cancer and Their Clinical Implications

The nuclear retinoic acid receptor may play a critical role in the process of lung carcinogenesis. Alteration or loss of nuclear retinoic acid receptors (RARs) has been associated with progression in premalignant and malignant tissues and it is associated with malignant transformation in human cells. Vitamin A derivates, such as retinoic acid, have emerged as adjuvant to therapy in several types of cancer with favorable effects. Retinoic acid regulates the expression of target genes through the binding and activation of RARs, inhibiting growth proliferation. Diverse studies have evaluated different retinoids alone or in combination with chemotherapy in lung cancer, from which results have been controversial with benefits observed only in the subpopulation with high levels of triglycerides. Additionally, several large randomized trials using retinoids to prevent tobacco-related cancer have failed; due to the latter the use of retinoids in clinical trials remains controversial. However they could reduce the risk of cancer development in non-smokers. There is evidence that retinoids have different effects on lung cancer; still the identification of biomarkers could determinate their benefits as preventive or therapy agents. This review describes the RAR alterations during the development of Non-Small Cell Lung Cancer and sets out the importance of several cancer treatments with retinoid compounds.

Saltatory remodeling of Hox chromatin in response to rostrocaudal patterning signals

Hox genes controlling motor neuron subtype identity are expressed in rostro-caudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains, controlled by rostro-caudal patterning signals that trigger rapid, domain-wide clearance of repressive H3K27me3 Polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid (RA) leads to activation and binding of RA receptors (RARs) to Hox1-5 chromatin domains, followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and FGF signals induce expression of Cdx2 transcription factor that binds and clears H3K27me3 from Hox1-9 chromatin domains, leading to specification of brachial/thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostro-caudal neural identity and that maintenance of these chromatin domains ensures transmission of the positional identity to postmitotic motor neurons later in development.

Identification of daidzein as a ligand of retinoic acid receptor that suppresses expression of matrix metalloproteinase-9 in HaCaT cells

Retinoids have been used as therapeutics for diverse skin diseases, but their side effects limit clinical usage. Here, we report that extracts of two soybeans, Glycine max and Rhynchosia nulubilis, and their ethyl acetate fractions increased the transcriptional activity of retinoic acid receptors (RARs), and that daidzin and genistin were the major constituents of the active fractions. Daidzin and its aglycone, daidzein, induced transcriptional activity of RAR and RARγ. FRET analysis demonstrated that daidzein, but not daidzin, bound both RAR and RARγ with EC50 values of 28μM and 40μM, respectively. Daidzein increased expression of mRNA of RARγ through direct binding of RAR and recruitment of p300 to the RARγ2 promoter. Further, mRNA and gelatinolytic activity of matrix metalloproteinase-9 were decreased by daidzein in HaCaT cells. Together, these results indicate that daidzein functions as a ligand of RAR that could be a candidate therapeutic for skin diseases.

Retinoic Acid Receptors Recognize the Mouse Genome through Binding Elements with Diverse Spacing and Topology

Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

Hepatocyte nuclear factor 4α (HNF4α) in coordination with retinoic acid receptors (RARs) increases all‐trans‐retinoic acid (RA)‐dependent CYP26A1 gene expression in hepatocytes

CYP26A1, a cytochrome P450, catalyzes exclusively the oxidative inactivation of all‐trans‐RA, the major hormonal metabolite of vitamin A. RA is a pan‐agonist of RARs, promoting RAR binding to DNA response elements of numerous genes. CYP26A1 is very highly induced by RA in the liver, generally to a greater extent than in other tissues, suggesting that a liver‐specific factor may be required for its physiological transcriptional response. In this study, we cotransfected HNF4α, with or without RARs, and demonstrated RA‐dependent activation of a human CYP26A1 promoter‐luciferase construct in HepG2 cells. Analysis of a 2.5‐kbp putative CYP26A1 promoter sequence identified 5 putative HNF4α DNA response elements: H1 located in a proximal region overlapping with an RAR element‐1 (RARE1); H2 and H3 in the distal region, close to RARE2 and RARE3; and H4 and H5 in intermediary regions. Chromatin immunoprecipitation analysis showed significantly increased binding of HNF4α and RARs in the proximal and distal CYP26A1 promoter regions in RA‐treated cells, as compared to the control. Mutational analysis of the individual HNF4α DNA‐response elements identified H1 as the major site for HNF4α binding because H1 mutation inhibited the promoter activity by ~90%, followed by H2 and H3 mutation each with ~50% inhibition. Therefore, our results indicate that HNF4α, as a liver‐enriched nuclear transcription factor, may coordinate with RARs to strongly induce CYP26A1 gene by RA, which may explain the high level of response to RA observed in the liver. (NIH CA‐90214)

Impact of intracellular alpha fetoprotein on retinoic acid receptors‐mediated expression of GADD153 in human hepatoma cell lines

The aim of our study was to gain a better understanding of the molecular mechanism underlying the previously unrecognized role of cytoplasmic alpha fetoprotein (AFP) in retinoic acid receptors (RAR) mediated expression and biological effects of GADD153. Using microarray analysis, the expression of the GADD153 gene showed the greatest fold change among apoptosis/growth related genes in response to ATRA. AFP was able to interact with RAR in HepG2 cells, which was undetectable in HLE cells owing to absence of AFP. ATRA promoted nuclei entrance of RAR, expression of GADD153 and apoptosis, and these changes were reversed after transfection with the afp gene or addition of AGN193109. The level of GADD153 was gradually elevated as the effect of AFP was counteracted by increasing dose or prolonging treatment time with ATRA in HepG2 cells. Knockdown of AFP in siRNA-transfected HepG2 cells or over-expression of AFP in afp gene-transfected HLE cells was synchronously associated with up-regulation or down-regulation, respectively, of GADD153 expression. Both ATRA administration and AFP knockdown were each able to promote greater binding of RAR to its response element with consequent elevation of the proportion of apoptotic cells. Conversely, transfection of HLE cells with pcDNA3.1-afp resulted in apparent reduction of RAR binding to DNA and change of biological effect. These data taken together demonstrate the involvement of AFP in RAR-mediated expression and biological effects of GADD153. These findings provide a novel insight into the mechanism underlying the impact of AFP on the RAR signal network.

A retinoic acid receptor RARα pool present in membrane lipid rafts forms complexes with G protein αQ to activate p38MAPK

Retinoic acid (RA) regulates several gene programs by nuclear RA receptors (RARs) that are ligand-dependent transcriptional transregulators. The basic mechanism for switching on transcription of cognate-target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes. In addition to these classical genomic effects, we recently demonstrated that RA also induces the rapid activation of the p38MAPK/MSK1 pathway, with characteristic downstream consequences on the phosphorylation of RARs and the expression of their target genes. Here, we aimed at deciphering the underlying mechanism of the rapid non-genomic effects of RA. We highlighted a novel paradigm in which a fraction of the cellular RARα pool is present in membrane lipid rafts, where it forms complexes with G protein alpha Q (Gαq) in response to RA. This rapid RA-induced formation of RARα/Gαq complexes in lipid rafts is required for the activation of p38MAPK that occurs in response to RA. Accordingly, in RA-resistant cancer cells, characterized by the absence of p38MAPK activation, RARα present in membrane lipid rafts does not associate with Gαq, pointing out the essential contribution of RARα/Gαq complexes in RA signaling.