Retinoid machinery in distinct neural stem cell populations with different retinoid responsiveness.
ABSTRACT: Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Rar?,?,?, Rxr?,?,?). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation.
Project description:Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.
Project description:To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (alpha, beta and gamma)-selective agonists and RARalpha- and RARgamma-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARalpha- and RARgamma-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARbeta-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 microM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARalpha antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARalpha- and RARgamma-selective retinoids. In contrast, the RARgamma antagonist only weakly inhibited RARalpha-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARgamma-selective retinoid. Our studies indicate that RARalpha is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARgamma isotype can also induce mucin genes. Furthermore these studies suggest that RARbeta is probably not (directly) involved in RA-induced mucin gene expression.
Project description:Retinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance.We applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets.We determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-?) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-?. Co-targeting of the retinoic acid and TGF-? pathways, through RA and kartogenin (KGN; a TGF-? signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells.Our approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-? signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.
Project description:BACKGROUND:Minocycline is a lipophilic tetracycline of increasing appeal in neuroscience as it inhibits microglial activation, a mechanism involved in numerous neuropsychiatric disorders. Own data point towards retinoid-mediated effects of minocycline in murine brain and skin, and towards a vicious cycle of neuroinflammation which is driven by microglial activation-induced breakdown of local retinoids such as retinoic acid (RA). We therefore sought to study minocycline's anti-inflammatory effects on human microglial-like monocyte-derived cells in the context of retinoid signaling. RESULTS:As hypothesized, minocycline exposure resulted in a substantial increase of RA levels in the human monocytic cell line THP-1. While pro-inflammatory stimulation with lipopolysaccharides resulted in increased tryptophane-degrading indoleamine-2,3-dioxygenase IDO-expression and TNF-? levels in primary human monocyte-derived microglial-like cells, this effect was attenuated by minocycline only in the presence of retinoids. The anti-inflammatory effects of minocycline on TNF-? expression were completely abolished by a pharmacological blockage of retinoic acid receptors (RARs) using BMS-493 and unaffected by selectively blocking retinoid-X-receptors using UVI-3003. CONCLUSIONS:Our data indicate for the first time a RA-dependent, anti-inflammatory effect for minocycline in human microglial-like cells via inhibition of local RA turnover. The RA-dependent mode of action for minocycline appears to be predominantly mediated through RAR-signaling.
Project description:Active vitamin A metabolites, known as retinoids, are essential for multiple physiological processes, ranging from vision to embryonic development. These small hydrophobic compounds associate in vivo with soluble proteins that are present in a variety of cells and in particular extracellular compartments, and which bind different types of retinoids with high selectivity and affinity. Traditionally, retinoid-binding proteins were viewed as transport proteins that act by solubilizing and protecting their labile ligands in aqueous spaces. It is becoming increasingly clear, however, that, in addition to this general role, retinoid-binding proteins have diverse and specific functions in regulating the disposition, metabolism and activities of retinoids. Some retinoid-binding proteins appear to act by sequestering their ligands, thereby generating concentration gradients that allow cells to take up retinoids from extracellular pools and metabolic steps to proceed in energetically unfavourable directions. Other retinoid-binding proteins regulate the metabolic fates of their ligands by protecting them from some enzymes while allowing metabolism by others. In these cases, delivery of a bound retinoid from the binding protein to the 'correct' enzyme is likely to be mediated by direct and specific interactions between the two proteins. One retinoid-binding protein was reported to enhance the ability of its ligand to regulate gene transcription by directly delivering this retinoid to the transcription factor that is activated by it. 'Channelling' of retinoids between their corresponding binding protein and a particular protein target thus seems to be a general theme through which some retinoid-binding proteins exert their effects.
Project description:The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte.
Project description:Retinoids regulate a wide spectrum of cellular functions from the embryo throughout adulthood, including cell differentiation, metabolic regulation, and inflammation. These traits make retinoids very attractive molecules for medical purposes. In light of some of the physicochemical limitations of retinoids, the development of drug delivery systems offers several advantages for clinical translation of retinoid-based therapies, including improved solubilization, prolonged circulation, reduced toxicity, sustained release, and improved efficacy. In this Review, we discuss advances in preclinical and clinical tests regarding retinoid formulations, specifically the ones based in natural retinoids, evaluated in the context of regenerative medicine, brain, cancer, skin, and immune diseases. Advantages and limitations of retinoid formulations, as well as prospects to push the field forward, will be presented.
Project description:Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARbeta2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARbeta2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARbeta2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARbeta2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARbeta2, DUSP6, and RGS16.
Project description:Retinoid treatment is employed during residual disease treatment in neuroblastoma, where the aim is to induce neural differentiation or death in tumour cells. However, although therapeutically effective, retinoids have only modest benefits and suffer from poor pharmacokinetic properties. In vivo, retinoids induce CYP26 enzyme production in the liver, enhancing their own rapid metabolic clearance, while retinoid resistance in tumour cells themselves is considered to be due in part to increased CYP26 production. Retinoic acid metabolism blocking agents (RAMBAs), which inhibit CYP26 enzymes, can improve retinoic acid (RA) pharmacokinetics in pre-clinical neuroblastoma models. Here, we demonstrate that in cultured neuroblastoma tumour cells, RAMBAs enhance RA action as seen by morphological differentiation, AKT signalling and suppression of MYCN protein. Although active as retinoid enhancers, these RAMBAs are highly hydrophobic and their effective delivery in humans will be very challenging. Here, we demonstrate that such RAMBAs can be loaded efficiently into cationic liposomal particles, where the RAMBAs achieve good bioavailability and activity in cultured tumour cells. This demonstrates the efficacy of RAMBAs in enhancing retinoid signalling in neuroblastoma cells and shows for the first time that liposomal delivery of hydrophobic RAMBAs is a viable approach, providing novel opportunities for their delivery and application in humans.
Project description:Resistance to therapeutic use of retinoids in glioma has been observed for over 20 years; however, the exact mechanism of resistance remains unknown. To understand retinoic acid resistance in glioma, we studied the turnover mechanism of retinoid receptor proteins in neural stem cells and glioma stem-like cells. Here, we show that in normal neural stem cells, proteasomal degradation of retinoid receptors involves sumoylation, ubiquitination and recognition by the valosin-containing protein (VCP/p97/Cdc48). We find that Sumo1 modification has a dual role to stabilize the retinoid receptor from unwanted degradation and signal additional modification via ubiquitination. Subsequently, the modified receptor binds to the VCP chaperone and both proteins are degraded by the proteasome. Additionally, we reveal that all trans retinoic acid (ATRA) induces VCP expression, creating a positive feedback loop that enhances degradation. In contrast, the pathway is impaired in the glioma stem-like cells resulting in the accumulation of sumoylated and high molecular weight forms of retinoid receptors that lack transcriptional activity and fail to be recognized by the proteasome. Moreover, modified receptor accumulation occurs before ATRA treatment; therefore, the transcritptional defect in glioma is due to a block in the proteasomal degradation pathway that occurs after the sumo modification step.