Akt phosphorylates and suppresses the transactivation of retinoic acid receptor alpha

The transactivation of nuclear receptors is regulated by both ligand binding and phosphorylation. We previously showed that RARalpha (retinoic acid receptor alpha) phosphorylation by c-Jun N-terminal kinase contributes to retinoid resistance in a subset of NSCLC cells (non-small cell lung cancer cells), but the aetiology of this resistance in the remainder has not been fully elucidated [Srinivas, Juroske, Kalyankrishna, Cody, Price, Xu, Narayanan, Weigel and Kurie (2005) Mol. Cell. Biol. 25, 1054-1069]. In the present study, we report that Akt, which is constitutively activated in NSCLC cells, phosphorylates RARalpha and inhibits its transactivation. Biochemical and functional analyses showed that Akt interacts with RARalpha and phosphorylates the Ser96 residue of its DNA-binding domain. Mutation of Ser96 to alanine abrogated the suppressive effect of Akt. Overexpression of a dominant-negative form of Akt in an NSCLC cell line decreased RAR phosphorylation, increased RAR transactivation and enhanced the growth-inhibitory effects of an RAR ligand. The findings presented here show that Akt inhibits RAR transactivation and contributes to retinoid resistance in a subset of NSCLC cells.     

Lymphocyte accumulation in the spleen of retinoic acid receptor-related orphan receptor gamma-deficient mice

The hormone nuclear receptor retinoic acid receptor-related orphan receptor gamma (RORgamma) plays important roles in thymocyte development and lymphoid organogenesis. RORgamma and its thymus-specific isoform RORgammat are expressed in the thymus, but not in the spleen and bone marrow (BM). However, RORgamma(-/-) mice have 2- to 3-fold more splenocytes than wild-type controls due to an accumulation of conventional resting B lymphocytes. The increase in B lymphocytes in RORgamma(-/-) mice is caused neither by abnormal B cell development in the BM nor by an obvious defect in the peripheral T cell compartment. Furthermore, analyses of BM chimeras using either RORgamma(-/-) or recombinase-activating gene-2(-/-) mice as recipients and wild-type or RORgamma(-/-) mice as donors, respectively, demonstrate that the splenic microenvironment of RORgamma(-/-) mice is defective, since wild-type T and B lymphocytes accumulated in these chimeric mice. In addition, T lymphocyte homeostasis was altered due to a lowered thymic output in RORgamma(-/-) mice. Collectively, these results suggest that RORgamma regulates lymphocyte homeostasis at multiple levels.     

Heme-oxygenase-mediated iron accumulation in the liver

Heme oxygenase (HO) isozymes, HO-1 and HO-2, catalyze the conversion of heme to iron, carbon monoxide, and biliverdin. The present study was aimed at elucidating the role of the HO system in iron accumulation and oxidative stress in the liver. We have also studied the regulation of an iron exporter, ferroportin-1 (FPN-1), as an adaptive response mechanism to increased iron levels. Sprague-Dawley rats were treated with HO inducer hemin or HO inhibitor tin-protoporphyrin IX (SnPPIX) for 1 month. A portion of liver tissues was subjected to RT-PCR for HO-1, HO-2, and FPN-1 gene expression as well as an HO activity assay. Paraffin-embedded tissues were stained for iron with Prussian blue. Hepatic iron concentration was measured by High Resolution-Inductively Coupled Plasma-Mass Spectrometry. 8-hydroxy-2'-deoxyguanosine (8-OHdG) stain, a sensitive and specific marker of oxidative DNA damage, was performed to assess oxidative stress. Hemin treatment led to augmented HO expression and activity in association with increased iron accumulation and oxidative stress. FPN-1 expression was also found to be upregulated. SnPPIX treatment reduced HO activity, intracellular iron levels, and oxidative stress as compared to controls. Our data provides evidence of increased HO activity as an important pro-oxidant mechanism leading to iron accumulation in the liver.     

Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats.

Retinoic acid (RA) and triiodothyronine (T3) exert many of their actions by binding to specific nuclear receptors (respectively, RA receptor (RAR) and T3) receptor (TR) belonging to a 'superfamily' of receptors. Some heterologous regulation of these receptors has been shown, and in particular regulation of the maximum binding capacity of TR by either retinol or RA. Now, using hyperthyroidic rats as a model, the effect of RA on binding capacity and on the mRNA levels of TR and RAR was investigated. The results show that the benefit of vitamin A treatment for the hyperthyroidic state, which has been described for a long time, could be the result of a down-heteroregulation of TR by RA, the active metabolite of retinol.     

Unbinding of retinoic acid from the retinoic acid receptor by random expulsion molecular dynamics

Unbinding pathways of retinoic acid (RA) bound to retinoic acid receptor (RAR) have been explored by the random expulsion molecular dynamics (REMD) method. Our results show that RA may exit the binding site of RAR through flexible regions close to the H1-H3 loop and beta-sheets, without displacing H12 from its agonist position. This result may explain kinetic differences between agonist and antagonist ligands observed for other nuclear receptors. The extended and flexible structure of RA initiated a methodological study in a simplified two-dimensional model system. The REMD force should in general be distributed to all atoms of the ligand to obtain the most unbiased results, but for a ligand which is tightly bound in the binding pocket through a strong electrostatic interaction, application of the REMD force on a single atom is preferred.     

All-trans retinoic acid suppresses exocrine differentiation and branching morphogenesis in the embryonic pancreas

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.     

A biologically active metabolite of retinoic acid from rat liver

1. Four major radioactive fractions have been isolated from the livers of vitamin A-deficient rats given [6,7-(14)C(2)]retinoic acid. 2. At least one of these was more potent than retinoic acid and approximately equal to retinol in the growth assay for vitamin A activity. 3. The biologically active material was chromatographically distinct from retinoic acid, retinol and retinal. 4. Alkaline hydrolysis of this material yielded an acidic compound containing all the radioactivity. 5. The methyl ester of the acidic product was unlike the methyl ester of retinoic acid in its chromatographic behaviour. 6. It is suggested that this metabolite may represent the active form of retinol in its growth-supporting role.     

Capsaicin suppresses liver fat accumulation in high-fat diet-induced NAFLD mice

ABSTRACT

Dietary capsaicin exhibits anti-steatosis activity in obese mice. High-fat diet (HFD)-induced mice is a highly studied approach to develop non-alcoholic fatty liver disease (NAFLD). In this study, we determined whether the topical application of capsaicin can improve lesions of NAFLD. The HFD-induced mice were treated with daily topical application of capsaicin for 8 weeks. Topical application of capsaicin reduced liver fat in HFD-fed mice. Capsaicin stimulated carnitine palmitoyl transferase (CPT)-1 and CD36 expression, which are associated with β-oxidation and fatty acids influx of liver while it decreased the expression of key enzymes involved in the synthesis of fatty acids, such as acetyl Co-A carboxylase (ACC) and fatty acid synthase (FAS). Immunohistochemical analysis revealed the elevated level of adiponectin in liver tissue of the capsaicin-treated mice. These results suggest that the topical application of capsaicin suppresses liver fat accumulation through the upregulation of β-oxidation and de novo lipogenesis in HFD-induced NAFLD mice.     

Cell type and gene-specific activity of the retinoid inverse agonist AGN 193109: divergent effects from agonist at retinoic acid receptor gamma in human keratinocytes.

Retinoids are important regulators of epithelial differentiation. AGN 193109 is a high-affinity antagonist and inverse agonist for the nuclear retinoic acid receptors (RARs). Paradoxically, both AGN 193109 and retinoid agonists inhibit the expression of the differentiation marker MRP-8 in normal human keratinocytes (NHKs). TTNPB, an RAR agonist, and AGN 193109 mutually antagonize MRP-8 inhibition at both mRNA and protein levels. We find that this antagonism, which is greatest at an AGN 193109:TTNPB ratio of about 10:1, is absent when either compound is in significant excess. The potent RARalpha-specific agonist, AGN 193836, has no effect on MRP-8 regulation. These data indicate that inverse agonists and agonists suppress MRP-8 in NHKs through RARgamma using distinct and mutually inhibitory mechanisms. The activity of AGN 193109 on MRP-8 is cell type specific. In differentiating ECE16-1 cervical cells, TTNPB inhibits while AGN 193109 induces MRP-8 mRNA levels. The effect of AGN 193109 on genes inhibited by retinoid agonists in NHKs is also selective; expression of the differentiation markers transglutaminase 1 and keratin 6 is not down-regulated by AGN 193109 whereas stromelysin-1 expression is suppressed. These results show a complex gene and cell context-specific interplay between agonist and inverse agonist for the regulation of gene expression.     

Design and synthesis of a potent,highly selective,orally bioavailable,retinoic acid receptor alpha agonist

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARβ, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARβ (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.     

A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency

Myeloproliferative syndromes (MPS) are a heterogeneous subclass of nonlymphoid hematopoietic neoplasms which are considered to be intrinsic to hematopoietic cells. The causes of MPS are largely unknown. Here, we demonstrate that mice deficient for retinoic acid receptor gamma (RARgamma), develop MPS induced solely by the RARgamma-deficient microenvironment. RARgamma(-/-) mice had significantly increased granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, and spleen. The MPS phenotype continued for the lifespan of the mice and was more pronounced in older mice. Unexpectedly, transplant studies revealed this disease was not intrinsic to the hematopoietic cells. BM from wild-type mice transplanted into mice with an RARgamma(-/-) microenvironment rapidly developed the MPS, which was partially caused by significantly elevated TNFalpha in RARgamma(-/-) mice. These data show that loss of RARgamma results in a nonhematopoietic cell-intrinsic MPS, revealing the capability of the microenvironment to be the sole cause of hematopoietic disorders.