Beneficial Effects Of Retinoic Acid Research Articles

The yin and yang of retinoic acid signaling in kidney diseases.

Retinoic acid (RA) signaling is involved in various physiological and pathological conditions, including development, tumorigenesis, inflammation, and tissue damage and repair. In kidneys, the beneficial effect of RA has been reported in multiple disease models, such as glomerulosclerosis, renal fibrosis, and acute kidney injury. In this issue of the JCI, Chen et al. report a pathway activated by RA signaling that is mediated by the retinoic acid receptor responder protein 1 (RARRES1). Specifically, RARRES1, which is proteolytically cleaved to release the extracellular domain, was endocytosed by podocytes to induce apoptosis and glomerular dysfunction kidney disease. These findings unveil the contrasting aspects, a Janus face, of RA signaling that may guide its therapeutic use.

Retinoic acid enhances the levels of IL-10 in TLR-stimulated B cells from patients with relapsing-remitting multiple sclerosis.

We have explored the beneficial effects of retinoic acid (RA) on B cells from multiple sclerosis (MS) patients. When co-stimulated via the toll-like receptors (TLRs) TLR9 and RP105, MS B cells secreted less of the anti-inflammatory cytokine interleukin 10 (IL-10) compared to B cells from healthy controls. Importantly, RA enhanced the secretion of IL-10 by MS-derived B cells without affecting the levels of the pro-inflammatory cytokine TNF-α. RA revealed the same ability to induce IL-10 as did interferon-β-1b (IFN-β-1b), and B-cells from patients treated with glatiramer acetate or IFN-β-1b still displayed the beneficial effects of RA on the IL-10/TNF-α ratio.

Retinoic acid modulates intrahippocampal levels of corticosterone in middle-aged mice: consequences on hippocampal plasticity and contextual memory

It is now established that vitamin A and its derivatives, retinoic acid (RA), are required for cognitive functions in adulthood. RA hyposignaling and hyperactivity of glucocorticoid (GC) pathway appear concomitantly during aging and would contribute to the deterioration of hippocampal synaptic plasticity and functions. Furthermore, recent data have evidenced counteracting effects of retinoids on GC signaling pathway. In the present study, we addressed the following issue: whether the stimulation of RA pathway could modulate intrahippocampal corticosterone (CORT) levels in middle-aged mice and thereby impact on hippocampal plasticity and cognitive functions. We firstly investigated the effects of vitamin A supplementation and RA treatment in middle-aged mice, on contextual serial discrimination task, a paradigm which allows the detection of early signs of age-related hippocampal-dependent memory dysfunction. We then measured intrahippocampal CORT concentrations by microdialysis before and after a novelty-induced stress. Our results show that both RA treatment and vitamin A supplementation improve “episodic-like” memory in middle-aged mice but RA treatment appears to be more efficient. Moreover, we show that the beneficial effect of RA on memory is associated to an increase in hippocampal PSD-95 expression. In addition, intrahippocampal CORT levels are reduced after novelty-induced stress in RA-treated animals. This effect cannot be related to a modulation of hippocampal 11β-HSD1 expression. Interestingly, RA treatment induces a modulation of RA receptors RARα and RARβ expression in middle-aged mice, a finding which has been correlated with the amplitude of intrahippocampal CORT levels after novelty-induced stress. Taken together, our results suggest that the preventive action of RA treatment on age-related memory deficits in middle-aged mice could be, at least in part, due to an inhibitory effect of retinoids on GC activity.

254 9-cis RETINOIC ACID INHIBITS CUMULUS CELL APOPTOSIS DURING IN VITRO MATURATION OF BOVINE OOCYTES THROUGH INHIBITION OF AP-1 PATHWAY

Cumulus cells (CC) play a critical role in oocyte maturation and fertilization via gap junctions. The oocyte itself maintains CC health to favour oocyte maturation via the secretion of paracrine growth factors. However, the antiapoptotic effects of oocyte-secreted factors follow a gradient from the site of the oocytes. Moreover, degrees of CC apoptosis are inversely related to the in vitro embryo development. Therefore, inhibition of CC apoptosis is important for efficient in vitro embryo development. The beneficial effects of retinoic acid (RA) during in vitro embryo production are well known in different species. However, the effect of RA on CC apoptosis is yet to be elucidated. All-trans RA and 9-cis RA are the natural components of retinoids, and all-trans RA are metabolized to 9-cis RA for physiological function. Therefore, the objective of the present study was to evaluate the effect of 9-cis RA on the mechanism for inhibition of apoptosis in CC. Slaughterhouse cumulus–oocyte complexes (COC) were matured in vitro in TCM-199-based in vitro maturation medium containing 0 or 5 mM 9-cis RA for 23 to 24 h (15 COC/100 μL droplet) at 38.5°C and 5% CO2 in air with maximum humidity. Following in vitro maturation, COC of a droplet were fixed in 4% paraformaldehyde for TUNEL staining using In Situ Cell Death Detection Kit (Roche, Budapest, Hungary). The proportion of apoptotic cells was estimated using Olympus Soft Imaging Solutions GmBH (Olympus, Münster, Germany). The COC of the remaining droplet were denuded. The CC were frozen and stored at –80°C. The CC of 3 different cultures were pooled, and total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA was reverse transcribed into cDNA using Omniscript Reverse Transcription kit (Qiagen). Relative expression of candidate genes was quantified using SYBER green real-time PCR with ΔΔ CT method. The expression was normalized against β-actin, glyceraldehyde 3-phosphate dehydrogenase, and 18s rRNA genes expression. The PCR efficiencies were calculated using relative calibration curves following 10-fold dilution series at 5 measuring points. Data were analysed for one-way ANOVA. The proportion of apoptotic cells was low in the 9-cis RA group (1.3 v. 3.3% of total CC; P < 0.05). Expression of tumor necrosis factor-α (11.1 v. 1.0; P < 0.001), caspase9 (2.0 v. 1.0; P < 0.01), and caspase3 (2.1 v. 1.0; P < 0.001) genes was down-regulated in the 9-cis RA group, whereas expression of Bcl2 gene was increased (1.0 v. 2.6 fold; P < 0.05). Moreover, the expression of c-fos gene of AP-1 pathway was down-regulated (1.9 v. 1 fold; P < 0.05) in the 9-cis RA group. Retinoic acid suppressed the expression of NF-kB, which in turn inhibits tumor necrosis factor-α-mediated caspase activity. However, the expression of NF-kB in CC was not affected by 9-cis RA (1.1 v. 1.0; P > 0.05). In conclusion, the present study indicated that 9-cis RA may inhibit cumulus cell apoptosis through suppression of AP-1 pathway. This work was partly supported by a scholarship from the BK21 program, the KRF (KRF-2008-211-F00011), the IPET (108068-03-1-SB010), and the KOSEF (10525010001-05N2501-00110).

Inhibitory effects of retinoic acid on invasiveness of human thyroid carcinoma cell lines in vitro

The prognosis of patients with metastasized thyroid carcinoma is not optimistic, necessitating the search for new treatment options. Beneficial effects of retinoic acid (RA) have been suggested in thyroid cancer differentiation and the present study was performed to investigate the anti-metastatic potential of RA in respect of important determinants of metastatic behavior in thyroid carcinoma, focusing on the role of invasion-associated proteins. Differentiated thyroid carcinoma cell lines FTC- 133 and XTC.UC1, and anaplastic thyroid cancer cell lines C643 and HTH74 were studied. All cell lines were cultured with alltrans- RA (ATRA) or the solvent ethanol. Invasion and adhesion potency in vitro was studied by transwell experiment and short-term adhesion assay. The involvement of invasion-associated proteins, urokinase type plasminogen activator (uPA), uPA receptor (uPAR), matrix metalloproteinase-2 (MMP-2) and E-cadherin, were investigated by semi-quantitative RT-PCR and Western blot. In vitro invasion assay revealed that ATRA treatment could reduce the invasive potency in all the thyroid cancer cell lines, with the most significant effect in anaplastic cancer cells. Short-term adhesion assay suggested that ATRA increases cancer cell adhesion to extracellular matrix (ECM) in C643, HTH74 and XTC.UC1, probably through a transcriptional and translational regulation of some attachment molecules. RT-PCR andWestern blot both revealed diminished expression of uPAR in all four carcinoma cell lines. In C643 and HTH74 cell lines, the expression of uPA was reduced and the expression of E-cadherin was increased, whereas the MMP-2 expression was not significantly down-regulated in ATRA-treated group. In ATRA-treated FTC-133 and XTC.UC1 cell lines, MMP-2 expression was decreased, but no significant changes in uPA and E-cadherin expression were observed. The present study demonstrates the influence of ATRA on both important determinants of metastatic behavior ("de-adhesion" and proteolysis) in thyroid carcinoma cell lines, especially in anaplastic cancer cells. These findings may add to the explanations for beneficial effects of RA in the treatment of metastatic thyroid carcinomas.

Beneficial effect of retinoic acid on the outcome of experimental acute renal failure.

Retinoic acid (RA) exerts beneficial effects on vascular remodelling and experimental nephritis, and plays a role in kidney development. Pathological changes caused by acute renal failure (ARF) result in high mortality. We determined whether RA ameliorates ARF-induced pathology caused by potassium dichromate (PD). Adult Wistar female rats (210-250 g) were randomly allocated to four groups: (i) an ARF group that received PD [15 mg/kg body weight (bw), single dose subcutaneously]; (ii) a group that received PD plus RA (1 mg/kg bw) beginning at 5 days before PD and that continued for 14 additional days; (iii) a group that received PD plus thyroxine (T(4); 8 micro g/100 g bw) with RA; and (iv) a group that received only the vehicle for PD (saline solution). We evaluated functional, biochemical and morphological characteristics of the kidneys. PD-induced alterations in serum creatinine, creatinine clearance (C(cr)) and fractional excretion of sodium (FeNa) were less severe when rats received RA. PD increased lipoperoxidation and this alteration was partially blocked by RA. Animals undergoing ARF showed severe histological injury (brush border loss, acidophilia, oedema, pyknosis, karyorhexis, cell detachment and disruption of the basement membrane). These alterations were less severe in RA-treated rats, indicating a protective effect on functional and morphological alterations. Alterations in urinary sediment were reduced by RA. The simultaneous administration of T(4) with RA did not produce additional protection. RA exerted beneficial effects on the duration and severity of renal damage induced by PD in a model of renal failure resembling ARF in humans. The protective effect of RA may be mediated by diminished lipoperoxidative damage.

235RETINOID-DEPENDENT MRNA EXPRESSION IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

As a transcription factor, retinoic acid (RA) can activate or silence a wide number of genes, thus inducing differentiation in cell systems and playing a role in cell cycle regulation. However, little is known of RA-dependent gene expression in the oocyte. Bovine oocytes and cumulus cells express most RA receptors, and the presence of 9-cis-RA during in vitro maturation (IVM) is beneficial to oocyte development (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). The present work analyzes the relative abundance of various developmentally important gene transcripts in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were manipulated in defined medium with polyvinyl-alcohol (DM-PVA). Those COCs undergoing prematuration were cultured for 24h in DM-PVA with 25μM roscovitine. For IVM, some prematured COCs were cultured for 24h in DM-PVA containing pFSH, LH and E2. Incubations were made at 39°C in an atmosphere of 5% CO2 in air and high humidity. Within experiments, COCs were cultured with nM 9-cis-RA 5, in 1% ethanol (both as vehicle and inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Using Real Time PCR (10 oocytes per group) (Rizos et al., 2003 Biol. Reprod. 68, 236) we examined the relative mRNA expression of genes involved in protection against free oxygen radicals (Mn-superoxide dismutase, MnSOD), glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH) and cell cycle events (Cyclin B1 and H1). Data (of 4 replicates) were analyzed by ANOVA and Duncan test (P<0.05). Regarding immature oocytes, prematuration in 1% ethanol increased cyclin B1 expression and decreased cyclin H1, while 9-cis-RA increased G6PDH. Maturation without additives increased cyclin B1 and G6PDH, but decreased cyclin H1 and MnSOD expression;; opposite trends were observed under increasing ethanol dosages (3% and 5%). Maturation with 1% ethanol or 9-cis-RA enhanced cyclin B1 and G6PDH, while reducing cyclin H1 and MnSOD expressions. The presence of 9-cis-RA during both prematuration and maturation processes tended to show more prominent effects than the ones observed when it was present only during prematuration or maturation alone. In our study, in presence of 9-cis-RA during both prematuration and maturation processes, the expression of cyclin B1 and G6PDH tended to increase, while cyclin H1 and MnSOD tended to decrease. However, the differences with the control group without additives were not significant. Our study during both prematuration and maturation processes show that beneficial effects of RA on oocyte developmental competence may not be related to the alteration of mRNA expression of the four genes analyzed. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175; 2003-05783).

325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP<0.01; a,bP<0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

Beneficial effects of retinoic acid on extracellular matrix degradation and attachment behaviour in follicular thyroid carcinoma cell lines.

The prognosis of patients with metastasised follicular thyroid carcinoma (FTC) is limited, necessitating the search for new treatment options. Beneficial effects of retinoids have been suggested in thyroid cancer and the present study was performed to investigate the effects of retinoic acid (RA) on important determinants of metastatic behaviour in FTC: the disengagement of tumour cells from the primary tumour and the degradation of extracellular matrix, focusing on the role of the plasmin activation system and the integrin and E-cadherin families of attachment molecules. Three FTC cell lines were studied: FTC-133, derived from the primary tumour; and FTC-236 and FTC-238, derived from metastases. FTC cell lines were cultured with 0.1, 1 and 10 microM 13-cis-RA or with the solvent DMSO for 1 and 5 days. Extracellular matrix degradation by these cell lines was studied by assessing the 48-h release of radioactivity from (35)S-methionine labelled extracellular matrix proteins synthesised by the MC3T3 cell line coated onto plastic. The involvement of constituents of the plasmin activation system was investigated by semi-quantitative RT-PCR and zymography. Attachment to extracellular matrix was studied by determining the number of adhering FTC cells to extracellular matrix coated onto plastic, 3 h after seeding. The involvement of attachment molecules was studied by RT-PCR with primers for integrin subclasses and E-cadherin and immunofluorescence for E-cadherin. Five days culturing with 10 microM RA reduced the degradation of extracellular matrix significantly in all cell lines: FTC-133 by 35%, FTC-236 by 74% and FTC-238 by 31%. Zymography revealed diminished activity of urokinase type plasminogen activator (uPA) in FTC-236 and FTC-238, but not in FTC-133 cultured with RA. mRNA expression of the uPA receptor was diminished in FTC-236. In the attachment assay, 10 microM RA for 5 days increased the number of adherent cells to extracellular matrix significantly by 91% in FTC-133, 64% in FTC-236 and 87% in FTC-238. No effects of RA on integrin or E-cadherin mRNA expression were observed. Immunofluorescence, however, revealed enhanced organisation of E-cadherin along the cell membrane by RA treatment. In conclusion, the present study demonstrates beneficial effects of RA on important determinants of metastatic behaviour in FTC cell lines, e.g. decreased degradation of extracellular matrix which may in part be explained by effects on the plasmin activation system and enhanced attachment to extracellular matrix. These findings may add to the explanations for beneficial effects of retinoids in thyroid cancer.

Dexamethasone does not counteract the response of acute promyelocytic leukaemia cells to all-trans retinoic acid.

Retinoic acid syndrome is a serious condition that may complicate the treatment of acute promyelocytic leukaemia patients. This syndrome may be treated effectively with high-dose corticosteroid therapy and, as a result, many patients with acute promyelocytic leukaemia receive dexamethasone at some point during treatment. We investigated whether dexamethasone would also antagonize the beneficial effects of retinoic acid. In t(15;17)-positive NB4 cells, dexamethasone did not affect the retinoic acid induced differentiation, normalization of PML-nuclear bodies or the induction of thrombomodulin mRNA. Finally, dexamethasone did not inhibit the anti-proliferative effect of retinoic acid but rather showed anti-proliferative activity itself.

Retinoids enhance the number of EGF receptors in corneal endothelial cells

Corneal endothelium appears to be an important target tissue for retinoids and epidermal growth factor (EGF). We report here that retinoic acid, or its synthetic analogue CBS-211 A (10 −8–10 −7 m) dose-dependently enhances the mitogenic effect of EGF on cultured bovine corneal endothelial cells (BCEC). Furthermore, retinoid treatment, especially with CBS-211 A, increases the EGF-binding capacity of BCEC without any modification of EGF receptor affinity. The addition of cycloheximide (0·5 μg ml −1) to cultures simultaneously with retinoids suppressed the retinoid effect on EGF-binding, whereas this protein synthesis inhibitor was ineffective when added 24 hr later. These results suggest that retinoids could induce the expression of EGF receptors on BCEC as an early event. This study, which demonstrates a cooperation between retinoids and EGF in corneal endothelium, could explain the beneficial effect of retinoic acid previously reported on this tissue repair in vivo. Our data could offer clues for new pharmaceutical strategies in ophthalmology therapy.

Retinoid-Induced Potentiation of Epidermal Growth Factor Mitogenic Effect on Corneal Endothelial Cells

Epidermal growth factor (EGF) is mitogenic for bovine corneal endothelial cells in culture. Pretreatment with either retinoic acid or the synthetic analog CBS-211 A ((E)-4-[2-(2-isopropyl-5-thienyl)propenyl]benzoic acid) at 10(-8)-10(-6) M enhanced the EGF effect. This potentiating effect of retinoids contrasts with their intrinsic activity, which results in cell growth inhibition in the absence of growth factor. The present data emphasize the previously reported beneficial effect of retinoic acid on corneal endothelium wound healing. This effect could be related to the potentiation of endogenous growth factors and further underlines the importance of retinoids in the medical treatment of corneal endothelial lesions.

Effects of locally applied retinoic acid on corneal xerophthalmia in the rat

Local application of retinoic acid in oil solution to the xerophthalmic cornea of the retinoldeficient rat cleared the lesion. Application of oil had no eurative effect. The xerophthalmic cornea is edematous and the beneficial effect of retinoic acid was assessed by comparing the weight of the retinoic-acid-treated cornea with that of the contralateral eye treated with oil. In 22 of 24 rats the cornea which had been treated with retinoic acid was lighter, i.e. less edematous, than the oil-treated cornea. A photographic record of the effect of retinoic acid confirmed these results. The question is discussed whether local treatment of the xerophthalmic cornea in vitamin A deficient and severely malnourished children would be beneficial.