Retinoic acid isomers produce malformations in postembryonic development of the Japanese flounder,Paralichthys olivaceus

We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.     

Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.     

Retinoic acid stimulates development of adult-type chromatophores in the flounder.

Premetamorphic flounder larvae were administered different doses of 9-cis retinoic acid (9cRA). 9cRA at 25 nM (the highest dose) not only stimulated adult-type (ad-) chromatophore development on the ocular (eyed) side, but also induced the development of ad-chromatophores on the blind (non-eyed) side of the metamorphosed fish. The ad-chromatophore development was stimulated by 9cRA only when administered to the larvae that were at late premetamorphosis and at early prometamorphosis. Ad-chromatophores actually appear much later, at the end of metamorphosis. 9cRA was not effective at later stages of metamorphosis. These results suggest that 9cRA stimulated the development or determination of the developmental fate of neural crest cells for chromatophores. The present results also suggest the presence of immature chromatophores or neural crest cells on both sides of the larval body of the flounder and that the ad-chromatophore development is somehow inhibited on the blind side in spontaneous metamorphosis. All-trans retinoic acid (atRA) had a similar effect on the ad-chromatophore development. In addition, both types of RAs affected the development of fin rays of the fish, resulting in deformity of fins when administered at high doses early in metamorphosis. The teratogenic effect of atRA was greater than that of 9cRA.     

Metabolism of a physiological amount of all-trans-retinol in the vitamin A-deficient rat

Because only retinol and not all-trans-retinoic acid (atRA) can satisfy all of the functions of vitamin A, we have investigated the retinol metabolites in tissues of vitamin A-deficient (VAD) rats responding to a radioactive dose of [20-(3)H]all-trans-retinol. As expected, atRA is the major vitamin A metabolite present in the target tissues of VAD rats given a physiological dose (1 microg) of [20-(3)H]all-trans-retinol (atROL). Both atROL and atRA were detected by high-performance liquid chromatographic (HPLC) analysis of the radioactivity extracted from the liver, kidney, small intestine, lung, spleen, bone, skin, or testis of these animals. Novel retinol metabolites were observed in the aqueous extracts from the testis, lung, and skin. However, these metabolites were detected in very small amounts and were not characterized further. Importantly, neither 9-cis-retinoic acid (9cRA), 9-cis-retinol (9cROL), nor 13-cis-retinoic acid (13cRA) was present in detectable amounts. The amounts of atRA varied in each tissue, ranging from 0.29 +/- 0.05 fmol of RA/g of tissue in the femurs to 12.9 +/- 4.3 fmol of RA/g of tissue in the kidneys. The absence of 9cRA in vivo was not due to degradation of this retinoid during the extraction procedure or HPLC analysis of the extracted radioactivity. As atROL completely fulfills all of the physiological roles of vitamin A, and 9cRA is not detected in any of the tissues analyzed, these results suggest that 9cRA may have no physiological relevance in the rat.     

Analysis, occurrence, and function of 9-cis-retinoic acid

Metabolic conversion of vitamin A (retinol) into retinoic acid (RA) controls numerous physiological processes. 9-cis-retinoic acid (9cRA), an active metabolite of vitamin A, is a high affinity ligand for retinoid X receptor (RXR) and also activates retinoic acid receptor (RAR). Despite the identification of candidate enzymes that produce 9cRA and the importance of RXRs as established by knockout experiments, in vivo detection of 9cRA in tissue was elusive until recently when 9cRA was identified as an endogenous pancreas retinoid by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This review will discuss the current status of the analysis, occurrence, and function of 9cRA. Understanding both the nuclear receptor-mediated and non-genomic mechanisms of 9cRA will aid in the elucidation of disease physiology and possibly lead to the development of new retinoid-based therapeutics. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.     

Retinoic acid stimulates the cell cycle machinery in normal T cells: involvement of retinoic acid receptor-mediated IL-2 secretion

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27(Kip1), increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.     

Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula

In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia.In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.     

The induction of prostaglandin E synthase and upregulation of cyclooxygenase-2 by 9-cis retinoic acid

9-cis Retinoic acid (9cRA) is a promising lead compound to design the retinoid X receptor (RXR) ligands with the ability to simultaneously activate RXR heterodimers with the selectivity to their nuclear receptor partners. In this study, we investigated the effects of 9cRA on the prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) production. 9cRA increased the PGE2 and TXA2 productions in the presence of lipopolysaccharide (LPS). All-trans retinoic acid, the retinoic acid receptor ligand, also increased their production. We revealed that cyclooxygenase (COX)-2 was clearly induced by 9cRA in the presence of LPS. The induction was not suppressed by indomethacin, which completely inhibited the increase in the LPS-stimulated prostanoid production by 9cRA. The expression levels of the toll-like receptor 4 and CD14, which were components of the LPS receptor complex, were increased by 9cRA in the presence and absence of LPS. PGE synthase was also clearly increased by 9cRA in the presence and absence of LPS. In this study, we noted that 9cRA increased the production of PGE2 and TXA2 by the induction of COX-2 and PGE synthase in the presence of LPS. The induction of the LPS receptor complex by 9cRA is able to upregulate the induction of COX-2 by LPS.     

Insulin-like growth factor binding protein-3 prevents retinoid receptor heterodimerization: implications for retinoic acid-sensitivity in human breast cancer cells

Insulin-like growth factor binding protein-3 (IGFBP-3) has both IGF-dependent and -independent effects on cell growth, which are frequently growth-inhibitory. Interestingly, the development of a more aggressive phenotype in breast cancer cells (BCCs) correlates positively with elevated expression of IGFBP-3 and is often associated with all-trans-retinoic acid (atRA)-resistance. IGFBP-3 was previously demonstrated to interact directly with retinoid X receptor (RXR). In this study we have shown that IGFBP-5 also interacts with RXR and that both IGFBPs interact with retinoic acid receptor (RAR). To investigate whether the presence of IGFBP-3 regulates breast cancer cell responsiveness to atRA, we immuno-neutralized the IGFBP-3 expressed by the atRA-resistant Hs578T and MDA-MB-231 BCCs (which express IGFBP-3 constitutively) and showed that they become more sensitive to the growth-inhibitory effects of atRA. Similarly, in Hs578T cells expressing a reporter gene under the control of an RAR response element (RARE), depletion of IGFBP-3 resulted in the induction of reporter gene expression in response to atRA. In investigating possible mechanisms for IGFBP-3 regulation of atRA-sensitivity, we found that IGFBP-3 blocked the formation of RAR:RXR heterodimers and disrupted the ligand-inducible receptor complex. Thus, IGFBP-3 has the potential to reduce the RARE-mediated transactivation of target genes and modulate the atRA-response in BCCs.     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.     

The novel retinoid AHPN/CD437 induces a rapid but incomplete apoptotic response in human myeloma cells

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.