Induction of histone acetylation on the CRBPII gene in perinatal rat small intestine

The expression of genes associated with lipid and vitamin A metabolism is elevated when the small intestinal mucosa is maturing rapidly during the perinatal period. We have previously reported that cellular retinol-binding protein type II (CRBPII) mRNA levels rise abruptly in the rat small intestine during this period. In this study, we examined whether the acetylation of histones H3 and H4 is involved in the intestinal expression of CRBPII during the perinatal stage. The expression of cyclin D1 and cyclin B1 genes, which are markers of cell proliferation, decreased markedly during the perinatal period, whereas expression of CRBPII as well as villin, a marker of intestinal maturation, increased rapidly. Using a ChIP assay, we showed rapid induction of acetylation of the histones H3 and H4 which interacted with the promoter/enhancer region of the CRBPII gene at this time. The binding of CBP and p300, which have histone acetyltransferase activity, as well as binding of retinoid X receptor alpha (RXRalpha) increased on the CRBPII promoter/enhancer region during the perinatal period. These results suggest that CRBPII gene expression during the perinatal period is associated with abrupt acetylation of histones H3 and H4 followed by the binding of CBP/p300 and RXRalpha.     

Developmental changes in gene expressions of beta-carotene cleavage enzyme and retinoic acid synthesizing enzymes in the chick duodenum

Vitamin A is derived from provitamin A carotenoids, mainly beta-carotene, by beta-carotene 15,15'-monooxygenase (BCMO1; EC 1.13.11.21). We previously reported that chick duodenal BCMO1 activity increased abruptly just after hatching. In this study, we further investigated mechanisms and physiological roles of the postnatal induction of BCMO1 expression in the chick duodenum. We showed that BCMO1 mRNA levels increased in the chick duodenum during postnatal period after hatching, but remain unchanged in the chick liver throughout the perinatal period. Serum hydrocortisone (HC) levels were also increased after hatching. Moreover, HC-administered chicks showed an enhancement of duodenal BCMO1 mRNA during the perinatal period. We further analyzed the developmental gene expression patterns of three types of retinoic acid (RA) synthesizing enzymes in the chick duodenum. Among them, retinal dehydrogenase 1 (RALDH1) mRNA levels in the chick duodenum increased during the postnatal period, indicating a similar developmental expression pattern to that of BCMO1. These results suggest that the postnatal induction of BCMO1 gene expression in the chick duodenum may be caused by the elevation of serum HC levels and may contribute to the RALDH1-mediated RA synthetic pathway.     

Expression of retinaldehyde dehydrogenase enzymes in mucosal dendritic cells and gut-draining lymph node stromal cells is controlled by dietary vitamin A

The vitamin A metabolite retinoic acid (RA) plays a crucial role in mucosal immune responses. We demonstrate in this study that RA-producing retinaldehyde dehydrogenase (RALDH) enzymes are postnatally induced in mesenteric lymph node (MLN) dendritic cells (DCs) and MLN stromal cells. RALDH enzyme activity in lamina propria-derived CD103(+) MLN-DCs did not depend on TLR signaling. Remarkably, RA itself could directly induce RALDH2 in both DCs and stromal cells in vitro. Furthermore, upon provision of a vitamin A-deficient diet, it was found that RA-mediated signaling was strongly reduced within the small intestines, while RALDH2 mRNA and RALDH enzyme activity in lamina propria DCs and MLN-DCs, as well as RALDH2 mRNA expression in MLN stromal cells, were strongly diminished. Moreover, supply of vitamin A to vitamin A-deficient mice restored RA-mediated signaling in the intestine and RALDH activity in lamina propria-derived CD103(+) MLN-DCs. Our results show that RA-dependent signaling within the intestine is indispensable for RALDH activity in the draining MLN.     

F9 embryocarcinoma cells: a cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling

Mouse F9 embryocarcinoma (EC) cells constitute a well established cell-autonomous model system for investigating retinoid signaling in vitro as, depending on culture conditions, retinoic acid (RA) can induce their differentiation into either primitive, parietal or visceral extraembryonic endoderm-like cells. These RA-induced differentiations are accompanied by decreases in proliferation rates, modifications of expression of subsets of RA-target genes, and induction of apoptosis. To elucidate the roles played by the multiple retinoid receptors (RARs and RXRs) in response to RA treatments, F9 EC cells lacking one or several RARs or RXRs were engineered through homologous recombination. Mutated RARs and/or RXRs were then reexpressed in given RAR or RXR null backgrounds. WT and mutant cells were also treated with different combinations of ligands selective for RXRs and/or for each of the three RAR isotypes. These studies lead to the conclusion that most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, apoptosis and activation of expression of a number of genes) are transduced by RARgamma/RXRalpha heterodimers, whereas some other events (e.g. parietal differentiation) are mediated by RARalpha/RXRalpha. heterodimers. They also demonstrate that both AF-1 and AF-2 activation functions of RARs and RXRs, as well as their phosphorylation, are differentially required in these RA-induced events. In RARgamma/RXRalpha heterodimers, the phosphorylation of RARgamma is necessary for triggering primitive differentiation, while that of RXRalpha is required for growth arrest. On the other hand, phosphorylation of RARalpha is necessary for parietal differentiation. Thus, retinoid receptors are sophisticated signal integrators that transduce not only the effects of their cognate ligands, but also those of ligands that bind to membrane receptors.     

Retinoids and retinoid-metabolic gene expression in mouse adipose tissues

Vitamin A and its analogs (retinoids) regulate adipocyte differentiation. Recent investigations have demonstrated a relationship among retinoids, retinoid-binding-protein 4 (RBP4) synthesized in adipose tissues, and insulin-resistance status. In this study, we measured retinoid levels and analyzed the expression of retinoid homeostatic genes associated with retinol uptake, esterification, oxidation, and catabolism in subcutaneous (Sc) and visceral (Vis) mouse fat tissues. Both Sc and Vis depots were found to contain similar levels of all-trans retinol. A metabolite of retinol with characteristic ultraviolet absorption maxima for 9-cis retinol was observed in these 2 adipose depots, and its level was 2-fold higher in Sc than in Vis tissues. Vis adipose tissue expressed significantly higher levels of RBP4, CRBP1 (intracellular retinol-binding protein 1), RDH10 (retinol dehydrogenase), as well as CYP26A1 and B1 (retinoic acid (RA) hydroxylases). No differences in STRA6 (RBP4 receptor), LRAT (retinol esterification), CRABP1 and 2 (intracellular RA-binding proteins), and RALDH1 (retinal dehydrogenase) mRNA expressions were discerned in both fat depots. RALDH1 was identified as the only RALDH expressed in both Sc and Vis adipose tissues. These results indicate that Vis is more actively involved in retinoid metabolism than Sc adipose tissue.     

Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells

Using genetic approaches in the mouse, we show that the primary target tissue of retinoic acid (RA) action during eye morphogenesis is not the retina nor the corneal ectoderm, which both express RA-synthesizing retinaldehyde dehydrogenases (RALDH1 and RALDH3), but the neural crest cell-derived periocular mesenchyme (POM), which is devoid of RALDH. In POM, the effects of the paracrine RA signal are mediated by the nuclear RA receptors heterodimers RXRalpha/RARbeta and RXRalpha/RARgamma. These heterodimers appear to control: (1) the remodeling of the POM through activation of Eya2-related apoptosis; (2) the expression of Foxc1 and Pitx2, which play crucial roles in anterior eye segment development; and (3) the growth of the ventral retina. We additionally show that RALDH1 and RALDH3 are the only enzymes that are required for RA synthesis in the eye region from E10.5 to E13.5, and that patterning of the dorsoventral axis of the retina does not require RA.     

Expression of retinaldehyde dehydrogenase (RALDH)2 and RALDH3 but not RALDH1 in the developing anterior pituitary glands of rats

Retinoic acid (RA) plays an important role in cell growth and tissue development and is also a regulating factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine hormone is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases catalyzing the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. In this study, we examined the expression of RALDH1, RALDH2, and RALDH3 mRNA in the rat embryonic pituitary gland. By in situ hybridization with digoxigenin-labeled cRNA probes, we detected mRNA expression for RALDH2 and RALDH3, but not RALDH1. The expression of RALDH2 and RALDH3 was located in Rathke’s pouch at embryonic day 12.5 (E12.5) and subsequently in the developing anterior pituitary gland. We also used quantitative real-time polymerase chain reaction to analyze RALDH2 and RALDH3 mRNA expression levels during the development of the pituitary gland. We found that pituitary RALDH2 and RALDH3 mRNA levels were high at E17.5 and decreased markedly after birth. Our study is the first to show that RALDH2 and RALDH3, but not RALDH1, are expressed in the embryonic anterior pituitary gland of the rat.     

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RARalpha/RXRalpha heterodimer to a novel retinoic acid response element in the promoter

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RARalpha/RXRalpha heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RARalpha/RXRalpha directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RARalpha/RXRalpha) in the induction of hiNOS by RA.