Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines

In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoidsall trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells.atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARα, RARβ, and RARγ were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARα does not mediate the growth stimulatory effects of retinoids, for a selective RARα antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth. RARα, RARβ, and RARγ mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line.     

An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.     

Analysis of the Factors Involved in the Retinoic Acid-Induced Differentiation of a Retinoid-Hypersensitive Embryonal Carcinoma Cell Mutant

Retinoic acid (RA) has been known to play an important role in cellular growth and differentiation as well as in vertebrate development. Many in vitro cell cultures also respond to RA by differentiating. Perhaps the most widely studied of these cultures are embryonal carcinoma (EC) cells. We have used an RA-hypersensitive EC cell mutant, created by retroviral insertion, to analyze the activity of the identifiable components in the RA response pathway. We have analyzed the mRNA expression patterns of the retinoic acid receptors (RARs) α, β, and γ, the retinoid X receptors (RXRs) α, β, and γ, and the cellular retinoic acid binding proteins (CRABPs) I and II. Our results indicate that CRABP I, RAR β, and RAR γ mRNAs are expressed differentially between parent and RA-hypersensitive mutant cells. All three messages are present at higher basal levels and at earlier times after RA addition in the mutant relative to parental cells. All other elements examined are equivalently expressed. Therefore analyses of the expression patterns of CRABPs, RARs, and RXRs in these RA-hypersensitive cells point to the probable importance of CRABP I, RAR β, and RAR γ in the RA induction pathway and also indicate that CRABP II and RXR γ are not likely to be critical elements in the early differentiative response of cells to RA.     

Involvement of retinoic acid nuclear receptors in retinoic acid-induced tissue transglutaminase gene expression in rat tracheal 2C5 cells.

The involvement of retinoic acid nuclear receptors (RARs) in the induction of tissue transglutaminase (TG) by retinoic acid in rat tracheal 2C5 cells was determined. The levels of RAR alpha and RAR beta were altered in 2C5 cells by transfection with RAR expression vectors. Increased expression of RAR alpha increased the induction of tissue TG by retinoic acid. In contrast, decreased RAR alpha expression, using an antisense RAR alpha expression vector, diminished the normal level of tissue TG induction caused by retinoic acid. Transfectants overexpressing RAR beta were also more responsive to retinoic acid for the induction of tissue TG, although the magnitude of TG induction was not as great as resulted from RAR alpha overexpression. These results indicate that the levels of the RAR alpha and RAR beta dictate the magnitude of tissue TG induction by retinoic acid.     

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.     

Retinoid X receptor-mediated transdifferentiation cascade in budding tunicates

In the budding tunicate, Polyandrocarpa misakiensis, retinoic acid (RA) applied to buds promotes transdifferentiation of somatic cells to form the secondary body axis. This study investigated the gene cascade regulating such RA-triggered transdifferentiation in tunicates. Genes encoding retinoic acid receptor (RAR) and retinoid X receptor (RXR) were induced during transdifferentiation, and they responded to all-trans RA or 13-cis RA in vivo, whereas 9-cis RA had the least effects, demonstrating differences in the ligand preference between budding tunicates and vertebrates. In contrast to RAR mRNA, RXR mRNA could induce transdifferentiation-related genes such as RXR itself, ERK, and MYC in an RA-dependent manner and also induced β-catenin (β-CTN) RA-independently when it was introduced in vitro into tunicate cell lines that do not express endogenous RAR or RXR. Small interfering RNA (siRNA) of RXR dramatically attenuated not only RXR but also ERK and β-CTN gene activities. An ERK inhibitor severely blocked wound healing and dedifferentiation. β-CTN siRNA suppressed morphogenesis and redifferentiation, similar to RXR siRNA. These results indicate that in P. misakiensis, the main function of RA is to trigger positive feedback regulation of RXR rather than to activate RAR for unlocking downstream pathways for transdifferentiation. Our results may reflect an ancient mode of RA signaling in chordates.     

Retinoic Acid Receptor-Dependent,Cell-Autonomous,Endogenous Retinoic Acid Signaling and Its Target Genes in Mouse Collecting Duct Cells

Background

Vitamin A is necessary for kidney development and has also been linked to regulation of solute and water homeostasis and to protection against kidney stone disease, infection, inflammation, and scarring. Most functions of vitamin A are mediated by its main active form, all-trans retinoic acid (tRA), which binds retinoic acid receptors (RARs) to modulate gene expression. We and others have recently reported that renal tRA/RAR activity is confined to the ureteric bud (UB) and collecting duct (CD) cell lineage, suggesting that endogenous tRA/RARs primarily act through regulating gene expression in these cells in embryonic and adult kidney, respectively.

Methodology/Principal Findings

To explore target genes of endogenous tRA/RARs, we employed the mIMCD-3 mouse inner medullary CD cell line, which is a model of CD principal cells and exhibits constitutive tRA/RAR activity as CD principal cells do in vivo. Combining antagonism of RARs, inhibition of tRA synthesis, exposure to exogenous tRA, and gene expression profiling techniques, we have identified 125 genes as candidate targets and validated 20 genes that were highly regulated (Dhrs3, Sprr1a, and Ppbp were the top three). Endogenous tRA/RARs were more important in maintaining, rather than suppressing, constitutive gene expression. Although many identified genes were expressed in UBs and/or CDs, their exact functions in this cell lineage are still poorly defined. Nevertheless, gene ontology analysis suggests that these genes are involved in kidney development, renal functioning, and regulation of tRA signaling.

Conclusions/Significance

A rigorous approach to defining target genes for endogenous tRA/RARs has been established. At the pan-genomic level, genes regulated by endogenous tRA/RARs in a CD cell line have been catalogued for the first time. Such a catalogue will guide further studies on molecular mediators of endogenous tRA/RARs during kidney development and in relation to renal defects associated with vitamin A deficiency.