Retinoic Acid Receptor Gene Expression Research Articles

FRI372 Retinoic Acids Attenuate The Endometriotic Cell Proliferation Through A Cell-specific Receptor Isoform

Abstract Disclosure: M. Izawa: None. Y. Azuma: None. F. Taniguchi: None. [Background] Endometriosis has been accepted as an estrogen-dependent, proliferative and inflammatory disease for over two decades, and the hormonal therapy targeting the reduction of estrogen has been employed as the first choice of treatment to reduce the endometriosis-associated symptoms. However, the limitation of therapy is becoming evident. In the hope of overcoming the limitation, we focused on retinoids, which have been known to elicit antiproliferative activity in breast tumors. Retinoic acid receptor (RAR) expression has been known to correlate with estrogen receptor expression. [Objective] As a first step to test the antiproliferative activity of retinoids in endometriotic lesions, we examined the expression profile of the retinoic acid receptor (RAR) in endometriotic cells. We then examined the antiproliferative activity of retinoids and the effect of estrogen on their activity. [Patients] Institutional Review Boards approved this project. We obtained informed consent from all patients. The chocolate cyst lining in the ovaries of patients with endometriosis was the source of endometriotic tissue. [Methods] Endometriotic cells: Stromal cells were collected from endometriotic tissues and used as endometriotic cells throughout the experiment. RAR gene expression: Single-stranded cDNA was prepared and subjected to PCR. Primer sets were designed using sequences in the UCSF genome browser. At the end of PCR cycles, the reaction mixture was subjected to electrophoresis on agarose gel. As an internal control, β-tubulin mRNA expression was assayed in parallel. Cell proliferation: Endometriotic cells were treated with retinoids in the presence or absence of estradiol (E2). All-trans retinoic acid (ATRA) and selective RAR modulators (SRARMs) were used. The number of viable cells was estimated using WST-8 assay. Ki-67 expression: Ki-67 expression was examined by Western blotting. As an internal control, β-tubulin expression was assayed in parallel. [Results] RAR mRNA expression: Transcripts of wild-type RARα, RARβ, RARγ, and their splice variants were demonstrated. These mRNAs were expressed almost at a comparable level: a truncated form of RARα without a DNA binding domain, a truncated form of RARβ without a transactivation domain at the N-terminal, and a form of RARγ with a distinct transactivation domain at N-terminal region. Effect of Retinoic acids on cell proliferation: The cell proliferation rate and Ki-67 expression were downregulated in the presence of ATRA. Among SRARMs tested, a selective RARγ modulator decreased cell proliferation. The antiproliferative activity of ATRA was attenuated in the presence of E2 in some cells.[Conclusion]We tested the antiproliferative activity of retinoids in endometriotic cells. Although the endometriotic cells responded to retinoids, the present finding suggested the diversity of cell types in endometriotic lesions. Presentation: Friday, June 16, 2023

Beta-carotene as a novel therapy for the treatment of “Autistic like behavior” in animal models of Autism

Autism-affected individuals are characterized by lower plasma oxytocin and its ectoenzyme regulator CD38. Oxytocin, a hypothalamic hormone secreted upon the release of CD38, plays a role in social behavior and bonding. All-trans retinoic acid is a potent inducer of CD38 and can be used as a novel therapeutic strategy in autism. We investigated the role of beta-carotene in rescuing autistic-like behavior in BALB/c and BTBR mice. Beta-carotene derivatives are preferred as they are neither toxic nor teratogenic. Beta-carotene at 0.1–5.0 mg/kg was administered orally to BALB/c and BTBR newborn mice on days 1–7. They were tested at age 2–3 months for five behavioral tests for “autism”; in addition, brain CD38, oxytocin, oxytocin receptor, Brain Derived Neurotrophic Factor (BDNF) and retinoic acid receptor gene expression, serum oxytocin levels, and neurological score were evaluated.Beta-carotene administered at birth significantly increased T-maze alternations and led to longer time spent with an unfamiliar mouse in the “three-chamber test” and less time spent in the empty chamber. Furthermore, enhanced activity in the open field test; increased time spent in the reciprocal social interaction test; decreased grooming and bedding behaviors; and enhanced brain CD38, oxytocin, oxytocin receptor, BDNF, retinoic acid gene expression, and serum oxytocin levels. No changes in neurological score were observed.Beta-carotene oral supplementation to BALB/c and BTBR mice at birth significantly reduced restricted and stereotyped behaviors and interests, increased social interactions and communication, CD38, and oxytocin, probably by enhancing brain neuroplasticity without toxicity. Thus, beta-carotene administered after birth to newborns of families predisposed to “autism” has the potential to prevent/ameliorate” autistic like behavior”. These results support further clinical studies.

Impact of Vitamin A Supplementation on RAR Gene Expression in Multiple Sclerosis Patients

Vitamin A and its derivatives have been shown to modulate the immune system via retinoic acid receptor (RAR). This study explored the impact of retinyl palmitate supplementation on RAR subtype gene expression in peripheral blood mononuclear cells (PBMCs) in multiple sclerosis (MS) patients. The study designed as a double-blind randomized clinical trial in which relapsing remitting multiple sclerosis patients were evaluated. Both groups received one capsule 50,000 IU vitamin D3 per 2 weeks and one intramuscular injection interferon beta-1a per week. The intervention group received one 25,000 IU retinyl palmitate capsule daily for 6 months and the placebo group received one placebo capsule daily. The PBMCs were isolated from participants and the expression level changes of RAR-α and RAR-γ genes were determined by real-time PCR. After supplementation, in the intervention group, the RAR-α gene expression level was significantly decreased compared to the placebo group (p = 0.03); however, the expression of RAR-γ gene did not significantly change (p = 0.10). These results show that vitamin A supplementation can significantly downregulate the expression of RAR-α gene in PBMCs of MS patients that suggest the presence of in vivo regulatory mechanisms for the action of vitamin A on the immune system.

Convergence of c AMP, TGF-β and retinoic acid signaling pathways in cells of the embryonic palate

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-β) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-β1, cyclic adenosine 3′, 5′-monophosphate (cAMP) and RA signaling systems. TGF-β1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-β mRNA in MEPM cells, though only TGF-β1 inhibited RA-induced RAR-β protein expression. Forskolin, but not TGF-β1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-β gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-β-responsive, element. Thus, a putative TGF-β Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-β promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.

Retinoic acid receptor gene expression in hepatocellular carcinoma

Retinoic acid receptor gene expression in hepatocellular carcinoma

Retinoic acid receptor gene expression in hepatocellular carcinoma

Retinoic acid receptor gene expression in hepatocellular carcinoma

Effects of retinoic acid and estrogens on oxytocin gene expression in the rat uterus: in vitro and in vivo studies

We and others have previously identified functional estrogen (E) and retinoic acid (RA) response elements in the human and rat oxytocin (OT) gene promoters. Whereas there is no direct evidence for a significant role of E or RA in the regulation of rat hypothalamic OT gene expression, we have recently demonstrated that in vivo adminstration of E strongly stimulates uterine OT gene expression. Here, we show that in vivo administration of RA similarly induces a significant increase in uterine OT gene expression. Moreover, we report that the E and RA effects are reproducible in vitro. Using short-term uterine organ explant cultures derived from 18-day pregnant rats, we found that E (50 nM) and RA (0.4 nM) increased OT mRNA levels 5.2- and 3-fold, respectively, suggesting a direct action of these agents on uterine OT gene expression. Finally, we analyzed uterine E and RA receptor gene expression during pregnancy. Using semi-quantitative Northern blot analysis, we found that mRNAs encoding the E receptor, the RA receptor α and RA receptor β are present in rat uterus and that their levels rise by 3.7-, 3.6- and 5.8-fold, respectively, between day 14 of gestation and term. Taken together, the data suggest that, at term, the rat uterus has an increased capacity to respond to E and RA, and that both agents may be involved in mediating the dramatic increase of OT mRNA accumulation observed in the uterus at term.

Postnatal rat lung retinoic acid receptor (RAR) mRNA expression and effects of dexamethasone on RAR β mRNA

Retinoids exert multiple effects upon lung differentiation and growth. Although the mechanisms involved are presently poorly understood, increasing evidence points to a central role of nuclear retinoic acid receptors (RAR). The purpose of this study was to determine RAR mRNA expression profile during postnatal alveolarization, compared with the expression in prenatal and adult rat lung, and to describe the effects of dexamethasone (DEX) and oxygen on postnatal lung RAR gene expression. Total RNA was isolated from lungs of Sprague-Dawley rats on prenatal day 19, on postnatal days 1, 3, 7, 10, and 14 of life, and from adults. One subgroup of littermate pups was treated with DEX daily for 3 or 7 days. In a second experiment, rats were exposed to room air or to 95% oxygen for 72 hours, and received either DEX or saline. Northern hybridization showed that the levels of all RAR subtypes in fetal lung were 45% or less of levels at postnatal day 1. The 3.7 kb RAR alpha transcript levels were lower than day 1 on days 10 and 14 (relative to day 1, day 10 = 0.54 +/- 0.05; day 14 = 0.54 +/- 0.08), but there was no change in a 2.7 kb RAR alpha transcript over this time period. By contrast, RAR beta mRNA levels were significantly higher at days 3, 10, and 14 compared with day 1 (day 3 = 1.79 +/- 0.19; day 10 = 1.41 +/- 0.14; day 14 = 1.53 +/- 0.05). Similarly, RAR gamma mRNA expression levels were higher on day 10 (1.45 +/- 0.09), but by day 14 there was no difference from day 1. Adult lung 3.7 kb RAR alpha, 2.7 kb RAR alpha, and RAR gamma were lower than day 1, but RAR beta was significantly greater (3.7 alpha = 0.52 +/- 0.05; 2.7 alpha = 0.49 +/- 0.26; gamma = 0.74 +/- 0.06; beta = 1.63 +/- 0.22). Treatment with DEX prevented the rise in RAR beta mRNA occurring on day 3 and significantly lowered (0.65 +/- 0.06) the amount of RAR beta mRNA in day 7 lung. Exposure of rat pups to oxygen caused an increase in RAR beta mRNA (1.21 +/- 0.03). DEX treatment again decreased RAR beta mRNA in both control (0.55 +/- 0.06) and oxygen-exposed pups (0.67 +/- 0.12). In addition, 2.7 kb RAR alpha mRNA was decreased with the combination of DEX and oxygen exposure (0.63 +/- 0.06). The differential gene expression profiles and the response to DEX and oxygen of the various members of the RAR family suggest that each subtype may have a specific role during the period of alveolarization in rat lung.

Retinoic acid regulates gene expression of retinoic acid receptors α,β and γ in F9 mouse teratocarcinoma cells

Abstract The regulatory effects of retinoic acid (RA) on retinoic acid receptor (RAR) α, β and γ mRNA were examined in the F9 mouse teratocarcinoma cells. Northern blot hybridization showed that RA regulated all three types of RAR gene expression. The different transcripts for each RAR α and β were differentially regulated by RA. The maximum induction of the 2.8 kb RAR α and 3.3 kb RAR β transcript levels by RA took longer than the induction of the 3.8 kb RAR α and 3.5 kb RAR β transcript levels. The data suggest that these transcripts originated from different promoters. Short term treatment (≤24 hours) of RA induced both 3.1 and 3.3 kb RAR γ transcripts. Long term treatment (> 24 hours) of RA resulted in the inhibition of 3.1 kb mRNA, whereas the 3.3 kb mRNA remained elevated. In addition, a new RAR γ transcript of 2.9 kb was induced. In contrast to RAR α and β, the effect on RAR γ gene expression was irreversible. Cycloheximide did not prevent the effect of RA on RAR gene expression, whereas actinomycin D totally abolished the RA effect on the expression of all three receptor genes. The data suggest that the biological effects of RA may be constrained or augmented by differential regulation of its own receptor gene expression.