RA Signaling Pathway Research Articles

Abstract 5452: AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells

Abstract Constitutively activated WNT-signaling promotes stem cell (SC) overpopulation which drives most colorectal cancer (CRC) development. It is not known, however, how WNT-signaling activity regulates different SC sub-populations. We previously reported that SC population size decreases as AXIN2 is knocked down, and increases when AXIN2 is overexpressed. Changes in cell cycle and apoptotic rate were also observed. Here, we present our recent results on changes between different SC subpopulations (ALDH+ vs. LGR5+) that occur due to AXIN2 modulation. We hypothesize that modulation of AXIN2 promotes differential expansion between different CRC SC subpopulations (ALDH+ and LGR5+). We observed that AXIN2 not only increases LGR5+ SC population size, but also expands the ALDH+ SC population to a greater extent as shown by an increased ALDH+-to-LGR5+ SCs ratio. This indicates that AXIN2, via WNT signaling, regulates SC population size, particularly ALDH+ SCs. Since the ALDH SC marker is also a key component in the retinoic acid (RA) signaling pathway, we surmised that increased AXIN2 would not only lead to elevated ALDH levels, but also upregulate the RA-signaling pathway components in CRC cells. To this end, we conducted NanoString mRNA profiling to study the effects of AXIN2 knockdown and overexpression in HT29 and SW480 CRC cells. The results showed that increased AXIN2 significantly up-regulated several key components of the RA-signaling pathway. Taken together, our results indicate that AXIN2 drives a shift in SC subpopulation sizes favoring the ALDH+ SCs. It also promotes the RA-signaling component expression - a pathway known to regulate SC differentiation. As a potential treatment strategy for CRC, therapeutically inducing high levels of AXIN2 could shift the balance between SC populations towards ALDH+ SCs, which may provide a way to target cancer SCs with retinoids to induce their differentiation. Citation Format: Chi Zhang, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5452.

Insights into vitamin A in bladder cancer, lack of attention to gut microbiota?

Vitamin A has long been associated with bladder cancer, and many exogenous vitamin A supplements, vitamin A derivatives, and synthetic drugs have been investigated over the years. However, the effectiveness of these strategies in clinical practice has not met expectations, and they have not been widely adopted. Recent medical research on intestinal flora has revealed that bladder cancer patients exhibit reduced serum vitamin A levels and an imbalance of gut microbiota. In light of the close relationship between gut microbiota and vitamin A, one can speculate that a complex regulatory mechanism exists between the two in the development and occurrence of bladder cancer. As such, further exploration of their interaction in bladder cancer may help guide the use of vitamin A for preventive purposes. During the course of this review, attention is paid to the influence of intestinal microbiota on the vitamin A metabolism and the RA signaling pathway, as well as the mutual promotion relationships between them in the prevention of bladder cancer, In addition, it emphasizes the importance of intestinal microbiota for bladder cancer prevention and treatment.

The role of BMP4 in adipose-derived stem cell differentiation: A minireview.

Bone morphogenetic protein 4 (BMP4) is a member of the transforming growth factor beta (TGF-β) superfamily of cytokines responsible for stem cells' commitment to differentiation, proliferation, and maturation. To date, various studies have utilized BMP4 as a chemical inducer for in vitro differentiation of human mesenchymal stem cells (MSCs) based on its potential. BMP4 drives in vitro differentiation of ADSC via TGF-β signaling pathway by interactions with BMP receptors leading to the activation of smad-dependent and smad-independent pathways. The BMP4 signaling pathways are regulated by intracellular and extracellular BMP4 antagonists. Extracellular BMP4 antagonist prevents interaction between BMP4 ligand to its receptors, while intracellular BMP4 antagonist shutdowns the smad-dependent pathways through multiple mechanisms. BMP4 proved as one of the popular differentiation factors to induce ADSC differentiation into cell from mesodermal origin. However, addition of all-trans retinoic acid is also needed in trans-differentiation of ADSC into ectodermal lineage cells. Suggesting that both BMP4 and RA signaling pathways may be necessary to be activated for in vitro trans-differentiation of ADSC.

Identification and Expression Pattern of cyp26b1 Gene in Gonad of the Chinese Tongue Sole (Cynoglossus semilaevis).

Simple SummaryIn fish, it is obvious that the asynchronous development of the gonads and sexual dimorphism limit the development of aquaculture, so the research into sex-differentiation and gonadal growth is very important. Due to the sexual reversal phenomenon (genetic females becoming phenotypic males), the Chinese tongue sole (Cynoglossus semilaevis) is a great model for investigating sex-differentiation. Herein, we report one gene involved in sex-differentiation and gonadal growth of the Chinese Tongue Sole. The gene cyp26b1 (cytochrome P450 family 26 subfamily b member 1) is a metabolizing Retinoic Acid (RA) enzyme. Since it regulates RA to control sex determination and differentiation, cyp26b1 is considered a critical part of mammals’ ovary-antagonizing and testis-determining downstream passageway of Sry (sex-determining region Y) and Sox9 (sry- box transcription factor 9). In fish, the related research is reported only on the Japanese flounder (Paralichthys olivaceus) and zebrafish (Danio rerio). In the current investigation, the identification and expression pattern of the cyp26b1 gene in the Chinese tongue sole suggested that cyp26b1 might impact sex-differentiation and gonadal development.As an RA-metabolizing enzyme, cyp26b1 has a substantial impact on RA-signaling pathways. The cyp26b1 gene from the Chinese tongue sole was cloned and identified in this investigation. The cyp26b1 ORF was 1536 bp in length and encoded a 512 amino acid protein. A quantitative real-time PCR (qPCR) indicated that the cyp26b1 expression is no significant sexual dimorphism in the gonads at the 80 days post-hatching (dph) stages. After 4 months post-hatching (mph), the expression of cyp26b1 showed sexual dimorphism and lower level of expression in the ovaries than in the testes. An in situ hybridization demonstrated that cyp26b1 mRNA was primarily located in the testis. Interestingly, the cyp26b1 mRNA probe was also detected in the ovaries. These results suggested that cyp26b1 participates in the sex-differentiation and gonadal development of the Chinese tongue sole.

An efficient method of inducing differentiation of mouse embryonic stem cells into primitive endodermal cells

Primitive Endoderm (PrE) is an extraembryonic structure derived from inner cell mass (ICM) in the blastocysts. Its interaction with the epiblast is critical to sustain embryonic growth and embryonic pattern. In this study, we reported a simple and efficient method to induce the differentiation of mouse Embryonic Stem Cells (mESCs) into PrE cells. In the process of ESC monolayer adherent culture, 1 μM atRA and 10 μM CHIR inducers were used to activate RA and Wnt signaling pathways respectively. After 9 days of differentiation, the proportion of PrE cells was up to 85%. Further studies indicated that Wnt signaling pathway acted as a switch that RA induces mESCs differentiation between SMC and PrE cell. In the presence of only RA signaling, mESCs adopted the fate of smooth muscle cells (SMCs); Simultaneous activation of the Wnt signaling pathway changed the differentiation fate of mESCs into PrE cells. This efficient induction method can provide new cellular resources and models for relevant studies of PrE.

Analysis of Liver Function in Retinoic Acid Receptor Gamma Liver Specific Knockout Mice

Non-Alcoholic Fatty Liver Disease (NAFLD) affects about one-third of the United States population and continues to grow in prevalence. Characterizing a spectrum of disease, NAFLD can range from simple steatosis to fibrosis and cirrhosis, which later can progress to hepatocellular carcinoma. Treatment options for this disease are currently limited to changing diet and exercise. Research efforts have been geared towards identifying and investigating potential targets for treatment of those with NAFLD. Via binding to retinoic acid, the biologically active metabolite of the micronutrient vitamin A (retinol), retinoic acid receptors (RARs α, β. γ) are known to regulate energy metabolism, and it has been shown that NAFLD leads to an altered/impaired state of vitamin A metabolism in the liver. In addition, our laboratory demonstrated that stimulation of the RA signaling pathway reduced hepatic steatosis in a High Fat Diet-induced NAFLD mouse model. Since RARγ is highly expressed in the liver, we are investigating the role of RARγ in NAFLD by characterizing the functional and molecular phenotypes of a murine liver-specific RARγ knockout. To date we have found that there is no difference in the vitamin A levels in the livers of these RARγ knockout mice. Through the characterization of this murine transgenic model, we will be able to determine the role of RARy in the liver, as well as to test new therapeutics that act via this receptor to potentially prevent/treat NAFLD.

Effects of vitamin A and retinoic acid on mouse embryonic stem cells and their differentiating progeny.

Embryonic development is controlled by retinoids, and one approach that has been used to investigate the mechanisms for retinoid actions in developmental processes has been to study the effects of adding retinoids to cultures of pluripotent embryonic stem cells (ESC). To date, most in vitro retinoid research has been directed at deciphering the actions of all-trans retinoic acid (atRA). atRA is a derivative of all-trans retinol (a.k.a. vitamin A, VA), which mammals can generate via an enzyme-catalyzed pathway. atRA's effects on development result from its (1) activation of receptor complexes (RARs and RXRs) in the nucleus which then bind to and activate RA response elements (RAREs) in genes and (2) its interactions with processes that are initiated in the cytoplasm. While much work has focused on the impact of atRA on cell differentiation, VA, itself, has been shown to exert effects on the maintenance of ESC identity that are not dependent upon classic RA-signaling pathways. In this chapter, we present results from our laboratory and others using well-documented approaches for investigating the effects of retinoids on the differentiation of ESC in vitro and introduce a novel method that uses chemically-defined growth conditions. The merits of each approach are discussed.

Esophageal Organoids from Human Pluripotent Stem Cells Delineate Sox2 Functions during Esophageal Specification

Tracheal and esophageal disorders are prevalent in humans and difficult to accurately model in mice. We therefore established a three-dimensional organoid model of esophageal development through directed differentiation of human pluripotent stem cells. Sequential manipulation of bone morphogenic protein (BMP), Wnt, and RA signaling pathways was required to pattern definitive endoderm into foregut, anterior foregut (AFG), and dorsal AFG spheroids. Dorsal AFG spheroids grown in a 3D matrix formed human esophageal organoids (HEOs), and HEO cells could be transitioned into two-dimensional cultures and grown as esophageal organotypic rafts. In both configurations, esophageal tissues had proliferative basal progenitors and a differentiated stratified squamous epithelium. Using HEO cultures to model human esophageal birth defects, we identified that Sox2 promotes esophageal specification in part through repressing Wnt signaling in dorsal AFG and promoting survival. Consistently, Sox2 ablation in mice causes esophageal agenesis. Thus, HEOs present a powerful platform for modeling human pathologies and tissue engineering.

RIP140/PGC-1α axis involved in vitamin A-induced neural differentiation by increasing mitochondrial function

Vitamin A deficiency and mitochondrial dysfunction are both associated with neural differentiation-related disorders, such as Alzheimer’s disease (AD) and Down syndrome (DS). The mechanism of vitamin A-induced neural differentiation and the notion that vitamin A can regulate the morphology and function of mitochondria in its induction of neural differentiation through the RIP140/PGC-1α axis are unclear. The aim of this study was to investigate the roles and underlying mechanisms of RIP140/PGC-1α axis in vitamin A-induced neural differentiation. Human neuroblastoma cells (SH-SY5Y) were used as a model of neural stem cells, which were incubated with DMSO, 9-cis-retinoic acid (9-cis-RA), 13-cis-retinoic acid (13-cis-RA) and all-trans-retinoic acid (at-RA). Neural differentiation of SH-SY5Y was evaluated by Sandquist calculation, combined with immunofluorescence and real-time polymerase chain reaction (PCR) of neural markers. Mitochondrial function was estimated by ultrastructure assay using transmission electron microscopy (TEM) combined with the expression of PGC-1α and NEMGs using real-time PCR. The participation of the RA signaling pathway was demonstrated by adding RA receptor antagonists. Vitamin A derivatives are able to regulate mitochondrial morphology and function, and furthermore to induce neural differentiation through the RA signaling pathway. The RIP140/PGC-1α axis is involved in the regulation of mitochondrial function in vitamin A derivative-induced neural differentiation.

A long range distal enhancer controls temporal fine-tuning of PAX6 expression in neuronal precursors

Proper embryonic development relies on a tight control of spatial and temporal gene expression profiles in a highly regulated manner. One good example is the ON/OFF switching of the transcription factor PAX6 that governs important steps of neurogenesis. In the neural tube PAX6 expression is initiated in neural progenitors through the positive action of retinoic acid signaling and downregulated in neuronal precursors by the bHLH transcription factor NEUROG2. How these two regulatory inputs are integrated at the molecular level to properly fine tune temporal PAX6 expression is not known. In this study we identified and characterized a 940-bp long distal cis-regulatory module (CRM), located far away from the PAX6 transcription unit and which conveys positive input from RA signaling pathway and indirect repressive signal(s) from NEUROG2. These opposing regulatory signals are integrated through HOMZ, a 94 bp core region within E940 which is evolutionarily conserved in distant organisms such as the zebrafish. We show that within HOMZ, NEUROG2 and RA exert their opposite temporal activities through a short 60 bp region containing a functional RA-responsive element (RARE). We propose a model in which retinoic acid receptors (RARs) and NEUROG2 repressive target(s) compete on the same DNA motif to fine tune temporal PAX6 expression during the course of spinal neurogenesis.

Negative interplay of retinoic acid and TGF-β signaling mediated by TG-interacting factor to modulate mouse embryonic palate mesenchymal-cell proliferation.

Mesenchymal-cell proliferation is the main process in shelf outgrowth. Both all-trans-retinoic acid (atRA) and transforming growth factor-β3 (TGF-β3) play an important role in mouse embryonic palate mesenchymal (MEPM) cell proliferation. In the present study, we investigated the crosstalk between RA and TGF-β signaling in MEPM-cell proliferation. We found that atRA inhibited MEPM-cell proliferation by downregulating TGF-β/Smad signaling and that TGF-β3 treatment was able to antagonize RA signaling. Transforming growth-interacting factor (TGIF) is a transcriptional repressor that suppresses both TGF-β- and retinoid-driven gene transcription. Furthermore, we investigated the role of TGIF in the interaction between both TGF-β and RA signaling in MEPM-cell proliferation. The results showed that both atRA and TGF-β3 significantly increased the expression level of TGIF, and TGIF mediated the negative interaction between TGF-β and RA signaling pathways, which depended on TGIF binding to Smad2 or RARβ (RA receptor beta). Moreover, after deletion of TGIF, both the effects of atRA on TGF-β-dependent protein expression and the effects of TGF-β on RA-dependent protein expression were lost. So we conclude that there is a negative functional interplay of RA and TGF-β signaling mediated by TGIF to modulate MEPM-cell proliferation.

Vitamin A deficiency impairs spatial learning and memory: the mechanism of abnormal CBP-dependent histone acetylation regulated by retinoic acid receptor alpha.

Vitamin A (VA) is an essential micronutrient. Numerous studies have confirmed that VA deficiency (VAD) leads to a decline in learning and memory function. Our previous studies have demonstrated that retinoic acid nuclear receptor α (RARα) in the hippocampus plays a crucial role in learning and memory, but the exact mechanism for this process is unclear. Epigenetic modifications, particularly histone acetylation, are involved in nervous system development, learning and memory function, and the pathogenesis of neurodegenerative diseases. Histone acetyltransferases (HATs), such as CREB-binding protein (CBP), E1A-binding protein p300 (p300), and p300/CBP-associated factor (PCAF), are critical for regulating memory function. The current study uses RARα and CBP as examples to study the connections between the RA signaling pathway and histone acetylation modification and to reveal the epigenetic mechanism in VAD-induced learning and memory impairment. This study examined the expression of RARα, HATs, acetylated histone H3/H4, and memory-related genes (Zif268, cFos, FosB), as well as the interaction of RARα and CBP in the hippocampus of 8-week-old rats. Additionally, the changes shown in vivo were further assessed in primary cultured neurons with the inhibition or overexpression of RARα. We found significantly lower levels of histone acetylation in the VAD rats. Furthermore, this downregulation, which impairs learning and memory, is induced by the dysregulation of CBP-dependent histone acetylation that is mediated by RARα. This work provides a solid theoretical foundation and experimental basis for the importance of ensuring sufficient nutritional VA during pregnancy and early life to prevent impairments of learning and memory in adulthood.

Vitamin A Deficiency Induces Congenital Spinal Deformities in Rats

Most cases of congenital spinal deformities were sporadic and without strong evidence of heritability. The etiology of congenital spinal deformities is still elusive and assumed to be multi-factorial. The current study seeks to elucidate the effect of maternal vitamin A deficiency and the production of congenital spinal deformities in the offsping. Thirty two female rats were randomized into two groups: control group, which was fed a normal diet; vitamin A deficient group, which were given vitamin A-deficient diet from at least 2 weeks before mating till delivery. Three random neonatal rats from each group were killed the next day of parturition. Female rats were fed an AIN-93G diet sufficient in vitamin A to feed the rest of neonates for two weeks until euthanasia. Serum levels of vitamin A were assessed in the adult and filial rats. Anteroposterior (AP) spine radiographs were obtained at week 2 after delivery to evaluate the presence of the skeletal abnormalities especially of spinal deformities. Liver and vertebral body expression of retinaldehyde dehydrogenase (RALDHs) and RARs mRNA was assessed by reverse transcription-real time PCR. VAD neonates displayed many skeletal malformations in the cervical, thoracic, the pelvic and sacral and limbs regions. The incidence of congenital scoliosis was 13.79% (8/58) in the filial rats of vitamin A deficiency group and 0% in the control group. Furthermore, vitamin A deficiency negatively regulate the liver and verterbral body mRNA levels of RALDH1, RALDH2, RALDH3, RAR-α, RAR-β and RAR-γ. Vitamin A deficiency in pregnancy may induce congenital spinal deformities in the postnatal rats. The decreases of RALDHs and RARs mRNA expression induced by vitamin A deprivation suggest that vertebral birth defects may be caused by a defect in RA signaling pathway during somitogenesis.

Retinoic acid synthesis and functions in early embryonic development

Retinoic acid (RA) is a morphogen derived from retinol (vitamin A) that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.

Spermatogonia Differentiation Requires Retinoic Acid Receptor γ

Vitamin A is instrumental to mammalian reproduction. Its metabolite, retinoic acid (RA), acts in a hormone-like manner through binding to and activating three nuclear receptor isotypes, RA receptor (RAR)α (RARA), RARβ, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (A(al)) spermatogonia, as well as during the transition from A(al) to A(1) spermatogonia, which is known to require RA; and 2) ablation of Rarg, either in the whole mouse or specifically in spermatogonia, does not affect meiosis and spermiogenesis but impairs the A(al) to A(1) transition in the course of some of the seminiferous epithelium cycles. Upon ageing, this phenomenon yields seminiferous tubules containing only spermatogonia and Sertoli cells. Altogether, our findings indicate that RARG cell-autonomously transduces, in undifferentiated spermatogonia of adult testes, a RA signal critical for spermatogenesis. During the prepubertal spermatogenic wave, the loss of RARG function can however be compensated by RARA, as indicated by the normal timing of appearance of meiotic cells in Rarg-null testes. Accordingly, RARG- and RARA-selective agonists are both able to stimulate Stra8 expression in wild-type prepubertal testes. Interestingly, inactivation of Rarg does not impair expression of the spermatogonia differentiation markers Kit and Stra8, contrary to vitamin A deficiency. This latter observation supports the notion that the RA-signaling pathway previously shown to operate in Sertoli cells also participates in spermatogonia differentiation.

Rab15 expression correlates with retinoic acid-induced differentiation of neuroblastoma cells

Neuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer deaths. Although retinoic acid (RA) is currently used to treat high-risk neuroblastoma patients in the clinic, RA-responsiveness is variable and unpredictable. Since no alterations in the RA-signaling pathway have been found in neuroblastoma cells, molecules correlated with RA-induced differentiation will provide predictive markers of RA-responsiveness for clinical use. The Rab family of small G proteins are key regulators of membrane traffic and play a critical role in cell differentiation and cancer progression. Although an increasing number of cancer-associated alternative splicing events have been identified, alternative splicing of Rab proteins remains to be characterized in neuroblastoma. In the present study, we focused on Rab15 that was originally identified as a brain-specific Rab protein and regulates the endocytic recycling pathway. We identified alternatively spliced Rab15 isoforms designated as Rab15CN and Rab15AN in neuroblastoma cells. Rab15CN was composed of 7 exons encoding 212 amino acids and showed brain-specific expression. Alternative splicing of exon 4 generated Rab15AN that was predicted to encode 208 amino acids and was predominantly expressed in testis. RA induced neuronal differentiation of neuroblastoma BE(2)-C cells and specifically up-regulated Rab15CN expression. Reciprocally, RA-induced differentiation was observed in Rab15CN-expressing BE(2)-C cells in preference to Rab15AN-expressing BE(2)-C cells. Furthermore, Rab15CN expression was also specifically up-regulated during RA-induced differentiation of newly established neuroblastoma cells from high-risk patients. These results suggest that Rab15 expression correlates with RA-induced differentiation of neuroblastoma cells.

Proinsulin C-peptide antagonizes the profibrotic effects of TGF-beta1 via up-regulation of retinoic acid and HGF-related signaling pathways.

Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.

Effect of 13-cis-retinoic acid on the genetic expression profile of human umbilical vein endothelial cells (HUVECs) determined by microarray

Treatment with isotretinoin (13-cis-retinoic acid; 13-cis RA) has been associated with hypertriglyceridemia and low levels of high-density lipoprotein (HDL)-cholesterol, which are well-established cardiovascular risk factors. The development of hypertriglyceridemia secondary to this treatment could predispose to the metabolic syndrome. Hence, because of the risk of atherosclerosis associated with this treatment and to help unravel the underlying mechanisms involved, we studied the effect of 13-cis RA on genetic expression in endothelial cells. Total RNA was isolated from human umbilical vein endothelial cells (HUVECs) treated without 13-cis RA (control) or incubated with 13-cis RA (1 microM) for 6 and 24 h. Microarray analysis (Affymetrix U133A array) with >22,000 probes was used with real time RT-PCR of a series of genes selected because of their involvement in the RA signaling pathway. The treatment of endothelial cells with 13-cis RA produced significant changes in the expression of genes implicated in cell adhesion and in lipid metabolism processes, specifically in the clearance of lipoprotein remnant particles and in HDL metabolism. Isotretinoin in the endothelium has an exacerbating effect on atherosclerosis risk factors. This is especially relevant in the regulation of expression of genes involved in the processes of cell adhesion and lipid metabolism.

NSPc1 is a cell growth regulator that acts as a transcriptional repressor of p21Waf1/Cip1 via the RARE element

The mammalian polycomb group proteins play an important role in cell cycle control and tumorigenesis. Nervous system polycomb 1 (NSPc1) is a newly identified transcription repressor, highly homologous with PcG protein Bmi-1. In this article, we showed that NSPc1 could promote tumor cell cycle progression and cell proliferation. Semi-quantitative RT–PCR showed that NSPc1 did not affect the expression levels of most Cyclin-depentent kinases (CDK) inhibitors except for p21Waf1/Cip1. Repression activity assays, chromatin immunoprecipitation (ChIP) and DNA pulldown assays all verified that NSPc1 represses the expression of p21Waf1/Cip1 by binding to the (−1357 to −1083) region of the p21Waf1/Cip1 promoter in vivo, and the repression effect is dependent on the retinoid acid response element (RARE element) within the above region of the p21Waf1/Cip1 promoter. Further analysis showed that NSPc1 could compete the RARE element site with RA receptors both in vitro and in vivo. Taken together, our results support the hypothesis that NSPc1 has a positive role in tumor cell growth by down-regulating p21Waf1/Cip1 via the RARE element, which directly connects transcriptional repression of PcGs to CDKIs and RA signaling pathways.

Functions of RARs and RXRs in vivo: Genetic dissection of the retinoid signaling pathway

Retinoids, the active metabolites of vitamin A, regulate complex gene networks involved in vertebrate morphogenesis, growth, cellular differentiation, and homeostasis. They are used for the treatment of skin disorders and as chemopreventive agents for certain cancers. Molecular biology and genetic studies performed during the last 15 years in vitro, using either acellular systems or transfected cells, have shown that retinoid actions are mediated through heterodimers between the 8 major RARα, β, and γ; isoforms and the 6 major RXRα, β and γ isoforms that belong to the nuclear receptor (NR) superfamily, and act as ligand-dependent transcriptional regulators. Furthermore, RXRs not only heterodimerize with RARs, but also with numerous other members of the NR superfamily. As in vitro studies are carried out under nonphysiological conditions, they only indicate what is possible, but not necessarily what is actually occurring in vivo. Therefore, mutations have been introduced by homologous recombination (HR) in F9 embryonal carcinoma (EC) cells, a cell-autonomous system that differentiates in the presence of RA, in order to disrupt RAR and RXR genes and establish their cellular and molecular functions in RA-induced differentiation. However, genetic approaches in the animal should be used to determine the function of retinoid receptors under truly physiological conditions. HR in embryonic stem (ES) cells, has therefore been used to generate null mutations of the various RARs and RXRs in the mouse germline. As reviewed here, the generation of such RAR and RXR germline mutations, combined with pharmacological approaches to block the RA signaling pathway, has provided many valuable insights on the developmental functions of RA receptors. However, due to (i) the complexity in "hormonal" signaling through transduction by the multiple RARs and RXRs, (ii) the functional redundancies (possibly artefactually generated by the mutations) within receptor isotypes belonging to a given gene family, and (iii) in utero or postnatal lethality of certain germline null mutations, these genetic studies through germline mutagenesis have failed to reveal many of the physiological functions of RARs and RXRs, notably in adults. We conclude that spatio-temporally controlled somatic mutations generated in animal models in given cell-types/tissues and at chosen times during pre- and postnatal life, are required to reveal the physiological and pathophysiological functions of the receptor genes involved in the retinoid signaling pathway throughout the life of the mouse.