TGFβ and Retinoic Acid Intersect in Immune-Regulation

Transforming growth factor (TGFβ) prevents TH1 and TH2 differentiation and converts naïve CD4 cells into Foxp3-expressing T regulatory (Treg) cell1, 2. In sharp contrast, in the presence of pro-inflammatory cytokines, including IL-6, TGFβ not only inhibits Foxp3 expression but also promotes the differentiation of pro-inflammatory IL17-producing CD4 effector T (TH17) cells3-5. This reciprocal TGFβ-dependent differentiation imposes a critical dilemma between pro- and anti-inflammatory immunity and suggests that a sensitive regulatory mechanism must exist to control TGFβ-driven TH17 effector and Treg differentiation. A vitamin A metabolite, retinoic acid (RA), was recently identified as a key modulator of TGFβ-driven immune deviation capable of suppressing TH17 differentiation while promoting Foxp3+Treg generation 6-10.     

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.     

Some 1,2-Diphenylethane Derivatives as Inhibitors of Retinoic Acid—Metabolising Enzymes

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50=52.1?μM; ketoconazole, 2.8?μM) and the RA-induced enzyme (100?μM, 65.9% inhibition; ketoconazole, 20?μM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.     

Synthesis of (±)-Tubaic Acid

An alternative synthesis of (±)-tubaic acid (I), a key intermediate compound of rotenone synthesis, has been accomplished by the Wittig reaction.     

Retinoic Acid Metabolism Inhibition by 3-Azolylmethyl-1H-indoles and 2, 3 or 5-(α-Azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or α-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 μM) ketoconazole exerted similar inhibitory effect (70% inhibition).     

α-LNA (α-D-Configured Locked Nucleic Acid)

Abstract

Two pyrimidine α-LNA nucleoside monomers have been synthesised and incorporated into α-configured oligonucleotides. A fully modified mixed α-LNA sequence displays unprecedented parallel stranded hybridisation with complementary RNA and a remarkable selectivity for RNA over DNA. Modelling shows α-LNA : RNA to form an extended duplex with a very broad major groove.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

Microarray analyses of gene expression are widely used, but reports of the same analyses by different groups give widely divergent results, and raise questions regarding reproducibility and reliability. We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes. These reports show substantial differences in their results. In this article, we review the methodology, results, and potential causes of differences in these applications of microarrays. Finally, we suggest practices to improve the reliability and reproducibility of microarray experiments.     

β-Transducin Repeat-containing Protein 1 (β-TrCP1)-mediated Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Protein Degradation Promotes Tumor Necrosis Factor α (TNFα)-induced Inflammatory Gene Expression

Cytokine modulation of the endothelium is considered an important contributor to the inflammation response. TNFα is an early response gene during the initiation of inflammation. However, the detailed mechanism by which TNFα induces proinflammatory gene expression is not completely understood. In this report, we demonstrate that silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) represses the expression of a subset of TNFα target genes in human umbilical vein endothelial cells. Upon TNFα stimulation, we observed an increase in the E3 ubiquitin ligase β-TrCP1 and a decrease in SMRT protein levels. We show that β-TrCP1 interacts with SMRT in a phosphorylation-independent manner and cooperates with the E2 ubiquitin-conjugating enzyme E2D2 to promote ubiquitination-dependent SMRT degradation. Knockdown of β-TrCP1 increases SMRT protein accumulation, increases SMRT association with its targeted promoters, and decreases SMRT target gene expression. Taken together, our results support a model in which TNFα-induced β-TrCP1 accumulation promotes SMRT degradation and the subsequent induction of proinflammatory gene expression.     

Promoter Methylation of the Retinoic Acid Receptor Beta2 (RARβ2) Is Associated with Increased Risk of Breast Cancer: A PRISMA Compliant Meta-Analysis

Background

Epigenetic studies demonstrate that an association may exist between methylation of the retinoic acid receptor beta2 (RARβ2) gene promoter and breast cancer onset risk, tumor stage, and histological grade, however the results of these studies are not consistent. Hence, we performed this meta-analysis to ascertain a more comprehensive and accurate association.

Materials and Methods

Relevant studies were retrieved from the PubMed, Embase and Chinese National Knowledge Infrastructure databases up to February 28, 2015. After two independent reviewers screened the studies and extracted the necessary data, meta-analysis was performed using Review Manager 5.2 software.

Results

Nineteen eligible articles, including 20 studies, were included in our analysis. Compared to non-cancerous controls, the frequency of RARβ2 methylation was 7.27 times higher in patients with breast cancer (odds ratio (OR) = 7.27, 95% confidence interval (CI) = 3.01–17.52). Compared to late-stage RARβ2 methylated patients, the pooled OR of early-stage ones was 0.81 (OR = 0.81, 95% CI = 0.55–1.17). The OR of low-grade RARβ2 methylated patients was 0.96 (OR = 0.96, 95% CI = 0.74–1.25) compared to high-grade RARβ2 methylated patients.

Conclusion

RARβ2 methylation is significantly increased in breast cancer samples when compared to non-cancerous controls. RARβ2 could serve as a potential epigenetic marker for breast cancer detection and management.     

Oncogenic Activity of Retinoic Acid Receptor γ Is Exhibited through Activation of the Akt/NF-κB and Wnt/β-Catenin Pathways in Cholangiocarcinoma

Aberrant expression and function of retinoic acid receptor γ (RARγ) are often involved in the progression of several cancers. However, the role of RARγ in cholangiocarcinoma (CCA), chemoresistant bile duct carcinoma with a poor prognosis, remains unclear. In the present study, we found that RARγ was frequently overexpressed in human CCA specimens. Its overexpression was associated with poor differentiation, lymph node metastasis, high serum carbohydrate antigen 19-9 level, and poor prognosis of CCA. Downregulation of RARγ reduced CCA cell proliferation, migration, invasion, and colony formation ability in vitro and tumorigenic potential in nude mice. RARγ knockdown resulted in upregulation of cell cycle inhibitor P21, as well as downregulation of cyclin D1, proliferating cell nuclear antigen, and matrix metallopeptidase 9, in parallel with suppression of the Akt/NF-κB pathway. Furthermore, overexpression of RARγ contributed to the multidrug chemoresistance of CCA cells, at least in part due to upregulation of P glycoprotein via activation of the Wnt/β-catenin pathway. Molecular mechanism studies revealed that RARγ interacted with β-catenin and led to β-catenin nuclear translocation. Taken together, our results suggested that RARγ plays an important role in the proliferation, metastasis, and chemoresistance of CCA through simultaneous activation of the Akt/NF-κB and Wnt/β-catenin pathways, serving as a potential molecular target for CCA treatment.