Systemic administration of the di-apocarotenoid norbixin (BIO201) is neuroprotective, preserves photoreceptor function and inhibits A2E and lipofuscin accumulation in animal models of age-related macular degeneration and Stargardt disease.

Atrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of Abca4-/- Rdh8-/- mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'-cis-norbixin (norbixin), a natural di-apocarotenoid, prepared from Bixa orellana seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in Abca4-/- Rdh8-/- mice of different ages, following three experimental designs: "preventive", "early curative" and "late curative" supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in Abca4-/- Rdh8-/- mice following the "early curative" supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment.

Visual Cycle Modulation as an Approach toward Preservation of Retinal Integrity.

Increased exposure to blue or visible light, fluctuations in oxygen tension, and the excessive accumulation of toxic retinoid byproducts places a tremendous amount of stress on the retina. Reduction of visual chromophore biosynthesis may be an effective method to reduce the impact of these stressors and preserve retinal integrity. A class of non-retinoid, small molecule compounds that target key proteins of the visual cycle have been developed. The first candidate in this class of compounds, referred to as visual cycle modulators, is emixustat hydrochloride (emixustat). Here, we describe the effects of emixustat, an inhibitor of the visual cycle isomerase (RPE65), on visual cycle function and preservation of retinal integrity in animal models. Emixustat potently inhibited isomerase activity in vitro (IC50 = 4.4 nM) and was found to reduce the production of visual chromophore (11-cis retinal) in wild-type mice following a single oral dose (ED50 = 0.18 mg/kg). Measure of drug effect on the retina by electroretinography revealed a dose-dependent slowing of rod photoreceptor recovery (ED50 = 0.21 mg/kg) that was consistent with the pattern of visual chromophore reduction. In albino mice, emixustat was shown to be effective in preventing photoreceptor cell death caused by intense light exposure. Pre-treatment with a single dose of emixustat (0.3 mg/kg) provided a ~50% protective effect against light-induced photoreceptor cell loss, while higher doses (1-3 mg/kg) were nearly 100% effective. In Abca4-/- mice, an animal model of excessive lipofuscin and retinoid toxin (A2E) accumulation, chronic (3 month) emixustat treatment markedly reduced lipofuscin autofluorescence and reduced A2E levels by ~60% (ED50 = 0.47 mg/kg). Finally, in the retinopathy of prematurity rodent model, treatment with emixustat during the period of ischemia and reperfusion injury produced a ~30% reduction in retinal neovascularization (ED50 = 0.46mg/kg). These data demonstrate the ability of emixustat to modulate visual cycle activity and reduce pathology associated with various biochemical and environmental stressors in animal models. Other attributes of emixustat, such as oral bioavailability and target specificity make it an attractive candidate for clinical development in the treatment of retinal disease.

AEG-1 regulates retinoid X receptor and inhibits retinoid signaling.

Retinoid X receptor (RXR) regulates key cellular responses such as cell growth and development, and this regulation is frequently perturbed in various malignancies, including hepatocellular carcinoma (HCC). However, the molecule(s) that physically govern this deregulation are mostly unknown. Here, we identified RXR as an interacting partner of astrocyte-elevated gene-1 (AEG-1)/metadherin (MTDH), an oncogene upregulated in all cancers. Upon interaction, AEG-1 profoundly inhibited RXR/retinoic acid receptor (RAR)-mediated transcriptional activation. Consequently, AEG-1 markedly protected HCC and acute myelogenous leukemia (AML) cells from retinoid- and rexinoid-induced cell death. In nontumorigenic cells and primary hepatocytes, AEG-1/RXR colocalizes in the nucleus in which AEG-1 interferes with recruitment of transcriptional coactivators to RXR, preventing transcription of target genes. In tumor cells and AEG-1 transgenic hepatocytes, overexpressed AEG-1 entraps RXR in cytoplasm, precluding its nuclear translocation. In addition, ERK, activated by AEG-1, phosphorylates RXR that leads to its functional inactivation and attenuation of ligand-dependent transactivation. In nude mice models, combination of all-trans retinoic acid (ATRA) and AEG-1 knockdown synergistically inhibited growth of human HCC xenografts. The present study establishes AEG-1 as a novel homeostatic regulator of RXR and RXR/RAR that might contribute to hepatocarcinogenesis. Targeting AEG-1 could sensitize patients with HCC and AML to retinoid- and rexinoid-based therapeutics.

Retinoid signaling is necessary for, and promotes long-term memory formation following operant conditioning.

Retinoic acid, a metabolite of vitamin A, is proposed to play an important role in vertebrate learning and memory, as well as hippocampal-dependent synaptic plasticity. However, it has not yet been determined whether retinoic acid plays a similar role in learning and memory in invertebrates. In this study, we report that retinoid signaling in the mollusc Lymnaea stagnalis, is required for long-term memory formation following operant conditioning of its aerial respiratory behaviour. Animals were exposed to inhibitors of the RALDH enzyme (which synthesizes retinoic acid), or various retinoid receptor antagonists. Following exposure to these inhibitors, neither learning nor intermediate-term memory (lasting 2 h) was affected, but long-term memory formation (tested at either 24 or 72 h) was inhibited. We next demonstrated that various retinoid receptor agonists promoted long-term memory formation. Using a training paradigm shown only to produce intermediate-term memory (lasting 2 h, but not 24 h) we found that exposure of animals to synthetic retinoids promoted memory formation that lasted up to 30 h. These findings suggest that the role of retinoids in memory formation is ancient in origin, and that retinoid signaling is also important for the formation of implicit memories, in addition to its previously demonstrated role in hippocampal-dependent memories.

ß-Cryptoxanthin uptake in THP-1 macrophages upregulates the CYP27A1 signaling pathway.

SCOPE: Mitochondrial sterol 27-hydroxylase (CYP27A1), a mediator of cholesterol homeostasis, is reported to exhibit antiatherogenic properties. Many studies suggested that all-trans retinoic acid can be used to treat atherosclerosis through retinoic acid receptor (RAR)-mediated upregulation of CYP27A1 expression. In this study, we hypothesized that ß-cryptoxanthin (ß-cry), as a natural ligand of RAR, might act as antiatherogenic agent by upregulating CYP27A1. METHODS AND RESULTS: We found that ß-cry treatment significantly upregulated genes involved in the uptake, transport, and metabolism of retinoids and the signaling pathway of CYP27A1 expression in THP-1 macrophages as detected by microarray analysis. Meanwhile, intracellular levels of ß-cry were correlated to the concentration and exposure time of the treatment. The expression of genes, involved in signaling pathway of CYP27A1, was dramatically decreased due to repressed activity of RAR. Higher level of 27-hydroxycholesterol was detected in ß-cry-treated macrophages by HPLC. Docking simulation showed that ß-cry could interact with cellular retinoic acid binding protein 2. These findings were further confirmed through microarray results. CONCLUSION: Our results provide strong evidence that ß-cry can be actively taken up by THP-1 macrophages and exhibits antiatherogenic effect on THP-1 macrophages by inducing CYP27A1 expression via RAR.

Synthesis of antioxidants for prevention of age-related macular degeneration.

Photooxidation of A2E may be involved in diseases of the macula, and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidant quercetin. These compounds contain water-solubilizing amine groups, and several were more potent inhibitors of A2E photooxidation than quercetin.

[RPE melanosomes bind A2E fluorophore of lipofuscin granules and products of its photooxidation].

The ability of melanosomes from human, bovine and frog retinal pigment epithelium cells (RPE) to bind A2E fluorophore of RPE lipofuscin granules and products of A2E photooxidation is investigated. RPE melanosomes are found to bind A2E molecules themselves as well as the molecules formed after A2E irradiation by visible light. In our experiments single melanosome was able to bind up to 0.08 fmol A2E. Antioxidant activity of melanosomes is compared to antioxidant activity of their complexes with A2E. It is shown by luminal chemiluminescence quenching in the presence of hydrogen peroxide that in A2E/melanosomes complex the chemiluminescence quenching is not significantly reduced. Comparison of inhibitory activity of melanosomes and their complexes with A2E on UV-induced (light conditions) and Fe(2+)-ascorbate-induced (dark conditions) peroxidation of photoreceptor outer segments (POS) demonstrated that bound A2E does not affect inhibitory ability of melanosomes in both systems. Thus, binding of A2E to RPE melanosomes in concentrations from 0.01 to 0.1 fmol A2E per melanosome does not significantly alter their antioxidant properties. It is supposed that both A2E and hydrophilic products of its photooxidation could be bound by RPE melanosomes and, thus, it lost the ability to exhibit toxic properties.

Inverse agonists and antagonists of retinoid receptors.

Nuclear receptors (NRs) are ligand-inducible transcription factors that regulate a plethora of cell biological phenomena, thus orchestrating complex events like development, organ homeostasis, immune function, and reproduction. Due to their regulatory potential, NRs are major drug targets for a variety of diseases, including cancer and metabolic diseases, and had a major societal impact following the development of contraceptives and abortifacients. Not surprisingly in view of this medical and societal importance, a large amount of diverse NR ligands have been generated and the corresponding structural and functional analyses have provided a deep insight into the molecular basis of ligand action. What we have learnt is that ligands regulate, via allosteric conformational changes, the ability of NRs to interact with different sets of coregulators which in turn recruit enzymatically active complexes, the workhorses of the ligand-induced epigenetic and transcription-regulatory events. Thus, ligands essentially direct the communication of a given NR with its intracellular environment at the chromatin and extragenomic level to modulate gene programs directly at the chromatin level or via less well-understood extranuclear actions. Here we will review our current structural and mechanistic insight into the functionalities of subsets of retinoid and rexinoid ligands that act generically as antagonists but follow different mechanistic principles, resulting in "classical" or neutral antagonism, or inverse agonism. In addition, we describe the chemical features and guidelines for the synthesis of retinoids/rexinoids that exert specific functions and we provide protocols for a number of experimental approaches that are useful for studies of the agonistic and antagonistic features of NR ligands.

All-trans retinoic acid ameliorates trinitrobenzene sulfonic acid-induced colitis by shifting Th1 to Th2 profile.

Inflammatory bowel disease is characterized with uncontrolled immune response in inflamed mucosa, with dominance of Th1 cells. Recently, all-trans retinoic acid has been shown that can lead T-cell response by suppressing Th17 development via retinoic acid receptor (RAR), but it is still unknown whether all-trans retinoic acid can modulate Th1 response of inflammatory bowel disease. In the experiment, we investigated the effect of all-trans retinoic acid on trinitrobenzene sulfonic acid (TNBS)-induced murine colitis, and the possible mechanism. Mice were intraperitoneally treated daily with all-trans retinoic acid (the agonist of RAR-alpha) or LE135 (the antagonist of RAR-alpha) or medium, and sacrificed 6 days later. Colon was collected for histological analysis and myeloperoxidase (MPO) activity measurement. Lamina propria mononuclear cells (LPMCs) were isolated, cultured, and assayed for the expressions of T-bet and GATA-3 by the use of Western blot and for cytokine levels by the use of ELISA. All-trans retinoic acid treatment inhibited inflammatory responses as shown by lower histological inflammatory scores and MPO activity, compared with LE135 and medium groups. Furthermore, in LPMCs culture supernatants, the levels of Th1 cytokines (INF-gamma, IL-12, and TNF-alpha) were decreased while those of Th2 cytokines (IL-4 and IL-10) were increased significantly in all-trans retinoic acid-treated mice. In addition, T-bet expression in LPMCs was inhibited and GATA-3 expression was up-regulated in all-trans retinoic acidtreated mice. On the contrary, LE135 showed the reverse effects in colon inflammation and cytokine profile. By shifting Th1 to Th2 profile in inflamed mucosa, all-trans retinoic acid down-regulates inflammatory response and ameliorates acute TNBS-induced colitis, which suggests the ligand of RAR-alpha-based pharmaceutical strategies for managing inflammatory bowel disease.

Tricyclic structures in medicinal chemistry: an overview of their recent uses in non-CNS pathologies.

Tricyclic compounds are sometimes considered as synonima of drugs healing central nervous system pathologies, although there are some well known examples of tricyclic derivatives marketed for different indications, such as antihistamines, antivirals and antiulceratives. Following the insertion of tricyclic structures in the "privileged structures" pool, several compounds bearing a central 7-membered ring and two aryl rings at its sides have been reported, and some of them have been progressed to advanced clinical trials. An overview of tricyclic derivatives reported in the literature since 1995, that are investigated for indications not directly related to central nervous system affections, shows the potential of these structures in a broad range of therapeutical indications, going from antiviral and anticancer compounds to the therapy of cardiovascular diseases. Very recent examples confirm the usefulness of tricyclic structures for the modern medicinal chemists.

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo.

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.

Protective effects of myricetin and related flavonols against A2E and light mediated-cell death in bovine retinal primary cell culture.

The present study was performed to investigate the effect of flavonols, namely myricetin and structurally related quercetin and kaempferol against A2E and blue light-induced photoreceptors death in primary retinal cell cultures. Primary retinal cell cultures were prepared from bovine retinas. Fourteen-day-old cultures were pretreated with different concentrations of myricetin, quercetin, kaempferol (1-40 microM) for 24 h, then treated with 30 microM of A2E or exposed to blue-actinic light for 20 h. Green nucleic acid stain assay was used to evaluate cell death. Photoreceptor and bipolar cells were immunolabeled with specific antibodies and were counted using automated microscope imaging and image-based cell counting software. Twenty hours exposure to blue light induced approximately 75% death of photoreceptors in bovine retinal cell cultures. Myricetin protected 100% of photoreceptors against blue-light-mediated damage with an EC(50) of 9+/-0.7 microM. Quercetin resulted in a maximum of 15% protection against light damage, and kaempferol was inactive. A2E induced photoreceptor and bipolar cell death in a concentration-dependent manner with EC(50) of 25 microM for photoreceptors and 31 microM for bipolar cells. Myricetin, quercetin and kaempferol protected against A2E-induced photoreceptors and bipolar cells death with EC(50) values of 2+/-0.3 microM, 2+/-0.3 microM, 5+/-0.09 microM and 0.8+/-0.07 microM, 0.44+/-0.06 microM, 1+/-0.4 microM, respectively. Caspase-3 inhibitor (Z-DEVD-fmk) protected 42% photoreceptors and 57% bipolar cells from A2E toxicity. In contrast, this inhibitor had no effect against light-induced photoreceptor damage. Despite the poor activity of quercetin and the inactivity of kaempferol against blue light, myricetin, quercetin and kaempferol exhibited approximately 100% protection against A2E toxicity. This suggests that light- and A2E-induced cell deaths are mediated through different pathways. These results suggest that myricetin functions as potent and effective neuroprotective agent for photoreceptor cells against A2E and light damage.

Lecithin:retinol acyltransferase is responsible for amidation of retinylamine, a potent inhibitor of the retinoid cycle.

Lecithin:retinol acyltransferase (LRAT) catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to all-trans-retinol (vitamin A) and plays an essential role in the regeneration of visual chromophore as well as in the metabolism of vitamin A. Here we demonstrate that retinylamine (Ret-NH2), a potent and selective inhibitor of 11-cis-retinal biosynthesis (Golczak, M., Kuksa, V., Maeda, T., Moise, A. R., and Palczewski, K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8162-8167), is a substrate for LRAT. LRAT catalyzes the transfer of the acyl group onto Ret-NH2 leading to the formation of N-retinylpalmitamide, N-retinylstearamide, and N-retinylmyristamide with a ratio of 15:6:2, respectively. The presence of N-retinylamides was detected in vivo in mice supplemented with Ret-NH2. N-Retinylamides are thus the main metabolites of Ret-NH2 in the liver and the eye and can be mobilized by hydrolysis/deamidation back to Ret-NH2. Using two-photon microscopy and the intrinsic fluorescence of N-retinylamides, we showed that newly formed amides colocalize with the retinyl ester storage particles (retinosomes) in the retinal pigment epithelium. These observations provide new information concerning the substrate specificity of LRAT and explain the prolonged effect of Ret-NH2 on the rate of 11-cis-retinal recovery in vivo.

A novel benzodiazepine selectively inhibits keratinocyte proliferation and reduces retinoid-induced epidermal hyperplasia in organ-cultured human skin.

Bz-423 is a new benzodiazepine that has cytotoxic and cytostatic effects against a number of cell types in culture, and recent studies have shown efficacy in experimental lupus models in rodents. The present study demonstrates that treating human skin in organ culture with Bz-423 suppresses retinoid-induced epidermal hyperplasia. Bz-423 suppresses hyperplasia in organ culture at concentrations that also inhibit keratinocyte proliferation in monolayer culture but that are not cytotoxic for keratinocytes and do not inhibit fibroblast growth. Under conditions in which keratinocyte proliferation is inhibited, there is no measurable effect on epidermal growth factor receptor activation, but there is reduced signaling at the level of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase. Suppression of keratinocyte growth by Bz-423 is associated with generation of intracellular oxidants. However, antioxidant treatment reduces keratinocyte cytotoxicity that occurs at high concentrations of Bz-423, but it does not inhibit growth suppression. Together, these data suggest that Bz-423 has the potential to limit the untoward effects associated with topical retinoid treatment, and in addition, may have therapeutic effects against other forms of epidermal hyperplasia.

Insulin-like growth factor binding protein-3 antagonizes the effects of retinoids in myeloid leukemia cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) can cause growth suppressive and proapoptotic effects on retinoids in many types of cancer cells. However, the expression and effects of IGFBP-3 in myeloid leukemia cells have not been elucidated. In this study, we found no IGFBP-3 expression in the human myeloid leukemia cell lines either at baseline or after stimulation with all-trans retinoic acid (ATRA). Human recombinant IGFBP-3 induced growth arrest and apoptosis of HL-60 and NB4 cells. We have previously identified RXR alpha as a nuclear receptor for IGFBP-3 and have proceeded to examine further the role of this interaction in leukemia cell lines. In signaling assays, IGFBP-3 potently suppressed RAR- and VDR-mediated signaling while enhancing RXR signaling. Interestingly, when IGFBP-3 was administered to these cells in combination with an RAR-selective ligand, the ability of these retinoids to induce differentiation was blunted. On the other hand, IGFBP-3 enhanced the effect of an RXR-selective ligand to induce differentiation of HL-60 and NB4 cells. Further studies showed that IGFBP-3 down-regulated (at the transcriptional level) the retinoid-induced expression of C/EBP epsilon in NB4 cells. Taken together, these results indicate that IGFBP-3 has antiproliferative activity against myeloid leukemia cells; while it enhances signaling through RXR/RXR, it blunts signaling by activated RAR/RXR.

Inhibition of disease progression by a novel retinoid antagonist in animal models of arthritis.

OBJECTIVE: To investigate the usefulness of a novel retinoic acid receptor (RAR) antagonist (BMS-189453) in animal models of arthritis. METHODS: BMS-189453 was tested in HIG-82 rabbit synovial fibroblasts to determine its ability to repress collagenase (matrix metalloproteinase-1, MMP-1) mRNA expression in vitro. Cells were stimulated with phorbol myristate acetate or interleukin 1 beta and mRNA quantified by slot-blot analysis. In vivo, BMS-189453 was evaluated in 2 animal models of arthritis: collagen induced arthritis (CIA) in mice and streptococcal cell wall induced arthritis (SCWA) in rats. Clinical scores for arthritis were recorded weekly. At the end of each study, limbs were evaluated histologically. In CIA, these results were correlated with mRNA levels for collagenase-3 (MMP-13) and stromelysin-1 (MMP-3) as determined by Northern blot. RESULTS: BMS-189453 reduced MMP-1 expression in HIG-82 synovial fibroblasts in culture. BMS-189453 treatment blocked the clinical progression of arthritis beyond soft tissue inflammation in the CIA model. In the SCWA model, BMS-189453 treatment resulted in significantly reduced swelling with no notable progression to joint distortion/destruction. Histological evaluation of the joints from animals in both models confirmed this result. Analysis of mRNA from the CIA paws showed that BMS-189453 prevented the overexpression of MMP-13 and MMP-3 in arthritic joints. CONCLUSION: Improvement in clinical and histologic variables in 2 separate animal models, along with simultaneous reduction in MMP expression in the affected joint, suggests that RAR antagonists such as BMS-189453 may be useful as agents to treat rheumatoid arthritis and for determining the role of MMP in disease progression. This is the first study to show the clinical potential of RAR antagonists in arthritis.

Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/Bax.

All-trans-retinoic acid and 9-cis-retinoic acid have been reported to have inhibitory effects on pancreatic adenocarcinoma cells and we have shown that this is partly due to induction of apoptosis. In this study, the mechanisms whereby 9-cis-retinoic acid induces apoptosis in these cells were investigated. An involvement of the Bcl-2 family of proteins was shown, such that 9-cis-retinoic acid causes a decrease in the Bcl-2/Bax ratio. Overexpression of Bcl-2 also resulted in inhibition of apoptosis induced by 9-cis-retinoic acid. Furthermore, two broad-range caspase inhibitors blocked DNA fragmentation induced by 9-cis-retinoic acid, but had no effect on viability defined by mitochondrial activity. Using synthetic retinoids, which bind selectively to specific retinoic acid receptor subtypes, we further established that activation of retinoic acid receptor-gamma is essential for induction of apoptosis. Only pan-retinoic acid receptor and retinoic acid receptor-gamma selective agonists reduced viability and a cell line expressing very low levels of retinoic acid receptor-gamma is resistant to the effects of 9-cis-retinoic acid. A retinoic acid receptor-beta/gamma selective antagonist also suppressed the cytotoxic effects of 9-cis-retinoic acid in a dose-dependent manner. This study provides important insight into the mechanisms involved in suppression of pancreatic tumour cell growth by retinoids. Our results encourage further work evaluating the clinical use of receptor subtype selective retinoids in pancreatic carcinoma.

Krüppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling.

We recently isolated a Krüppel-like zinc-finger transcription factor 5 (KLF5; also known as BTEB2 and IKLF), which is markedly induced in activated vascular smooth-muscle cells and fibroblasts. Here we describe our analysis of the in vivo function of KLF5 using heterozygous KLF5-knockout mice (Klf5(+/-)). In response to external stress, Klf5(+/-) mice showed diminished levels of arterial-wall thickening, angiogenesis, cardiac hypertrophy and interstitial fibrosis. Also, angiotensin II induced expression of KLF5, which in turn activated platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta (TGF-beta) expression. In addition, we determined that KLF5 interacted with the retinoic-acid receptor (RAR), that synthetic RAR ligands modulated KLF5 transcriptional activity, and that in vivo administration of RAR ligands affected stress responses in the cardiovascular system in a KLF5-dependent manner. KLF5 thus seems to be a key element linking external stress and cardiovascular remodeling.

Nicotine modulates the effects of retinoids on growth inhibition and RAR beta expression in lung cancer cells.

Epidemiological and animal studies have demonstrated that vitamin A and its natural and synthetic derivatives, retinoids, are effective agents in preventing the development of tobacco-associated cancers. Unfortunately, clinical trials of retinoids on cigarette smokers have shown lack of efficacy in preventing lung cancer. In our study, we investigated the effect of nicotine on the anti-cancer activity of all trans-retinoic acid (trans-RA) in human lung cancer cells. Our results demonstrated that nicotine could abrogate the growth inhibitory effect of trans-RA by suppressing its ability to induce the expression of RA receptor beta (RAR beta), a tumor suppressor. The inhibitory effect of nicotine was accompanied with induction of orphan receptor TR3. Inhibition of TR3 expression by overexpression of TR3 anti-sense RNA in H460 lung cancer cells strongly prevented the suppressive effect of nicotine on trans-RA activity. Treatment with nicotine or the cotransfection of TR3 expression vector inhibited the induction of RAR beta promoter activity by trans-RA in transient transfection assays. The inhibition of RAR beta promoter activity was due to the interaction of TR3 with orphan receptor COUP-TF, resulting in inhibition of COUP-TF DNA binding and transactivation on the RAR beta promoter. Furthermore, we found that nicotine failed to suppress the effect of a retinoid X receptor (RXR)-selective retinoid SR11237 on inducing both growth inhibition and RAR beta promoter activity, due to the ability of SR11237 to activate the RAR beta promoter through the RXR/TR3 heterodimer. Together, our results demonstrate that nicotine suppresses the growth inhibitory effects of trans-RA by inhibiting RAR beta expression through its induction of TR3 expression and suggest that RXR-selective retinoids may be more effective than classical retinoids for preventing and treating tobacco-associated cancers.

Oxidative stress-independent depletion of epidermal vitamin A by UVA.

In hairless mice, epidermal vitamin A (retinol and retinyl esters) is strongly decreased following a single exposure to UVB. Here, using the same mouse model, we studied the effects of UVA on epidermal vitamin A content, lipid peroxidation, and CRBP-I expression, as well as the putative prevention of vitamin A depletion or lipid peroxidation by topical alpha-tocopherol. An acute exposure to UVA completely depleted epidermal vitamin A with EC50 of 0.25 and 0.5 J per cm2 for retinyl esters and retinol, respectively; these values were 0.1 J per cm2 for both retinoids under UVB exposure. CRBP-I expression was increased 2-fold 8 h following UVA exposure (10 J per cm2), and this increase persisted for at least 16 h. A single UVA exposure induced a concentration-dependent epidermal lipid peroxidation (EC50 = 3.5 J per cm2) giving rise to 55.4 +/- 4.2 nmol lipid peroxides per g at 20 J per cm2, whereas UVB, up to 1 J per cm2, did not increase the basal concentration of 6.7 +/- 0.9 nmol lipid peroxides per g. On the other hand, topical menadione induced a concentration-dependent lipid peroxidation, but did not affect vitamin A content. Pretreatment with alpha-tocopherol (i) did not inhibit UV-induced vitamin A depletion, (ii) completely inhibited the increased lipid peroxidation induced by UVA or menadione, and (iii) accelerated reconstitution of epidermal vitamin A after UVB but not UVA induced depletion. Thus acute UVA induced both epidermal vitamin A depletion and lipid peroxidation, UVB induced only vitamin A depletion, and menadione induced only a lipid peroxidation; topical alpha-tocopherol prevented lipid peroxidation but not vitamin A depletion. These observations indicate (i) that CRBP-I neither provides protection to UVB- and UVA-induced epidermal vitamin A depletion, nor interferes significantly with reconstitution, and (ii) that the UV-induced vitamin A depletion and lipid peroxidation in mouse epidermis are unrelated processes. UV light does not destroy epidermal vitamin A through an oxidative stress but probably by a photochemical reaction in which UV radiations at about 325 nm give the corresponding activation energy.