Retinoic acid regulates the morphological development of sympathetic neurons

Interactions between all-trans-retinoic acid (RA) and bone morphogenetic proteins (BMPs) affect the expression of neurotrophin receptors in sympathetic neurons (Kobayashi et al., 1998). In this study, we examined the possibility that similar interactions might regulate the morphological development of these neurons. Under control conditions, embryonic rat sympathetic neurons formed axons but not dendrites; cells exposed to RA had a similar appearance. Profuse dendritic growth was observed upon exposure to BMP-7, and this was reduced by approximately 70% by RA. This inhibitory effect of RA was mediated primarily by retinoic acid receptors (RARs) and it exhibited substantial specificity because it was not associated with changes in either axonal elongation or cell survival. Moreover, mRNAs for enzymes required for synthesis of RA were expressed in the sympathetic neurons and retinoid activity was released from superior cervical ganglia. These observations suggest that retinoids may function as endogenous morphogens and regulate neural cell shape and polarity in developing sympathetic ganglia.     

Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in gamma-l-glutamyl-l-cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, gamma-glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that gamma-glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4-dependent expression of GSH1 and elevating the cellular levels of gamma-glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4-dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4-regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1-specific mechanisms exist that respond to GSH availability.     

Retinoic acid signaling regulates embryonic clock hairy2 gene expression in the developing chick limb

Embryo development proceeds under strict temporal control and an embryonic molecular clock (EC), evidenced by cyclic gene expression, is operating during somite formation and limb development, providing temporal information to precursor cells. In somite precursor cells, EC gene expression and periodicity depends on Retinoic acid (RA) signaling and this morphogen is also essential for limb initiation, outgrowth and patterning. Since the limb EC gene hairy2 is differentially expressed along the proximal-distal axis as growth proceeds, concomitant with changes in flank-derived RA activity in the mesenchyme, we have interrogated the role of RA signaling on limb hairy2 expression regulation. We describe RA as a positive regulator of limb hairy2 expression. Ectopic supplementation of RA induced hairy2 in a short time period, with simultaneous transient activation of Erk/MAPK, Akt/PI3K and Gli3 intracellular pathways. We further found that FGF8, an inducer of Erk/MAPK, Akt/PI3K pathways, was not sufficient for ectopic hairy2 induction. However, joint treatment with both RA and FGF8 induced hairy2, indicating that RA is creating a permissive condition for p-Erk/p-Akt action on hairy2, most likely by enhancing Gli3-A/Gli3-R levels. Finally, we observed an inhibitory action of BMP4 on hairy2 and propose a model whereby RA shapes limb hairy2 expression during limb development, by activating its expression and counteracting the inhibitory action of BMP4 on hairy2. Overall, our work reports a novel role for RA in the regulation of limb clock hairy2 gene expression and elucidates the temporal response of multiple intracellular pathways to RA signaling in limb development.     

Retinoic acid regulates endothelial cell proliferation during vasculogenesis

A dietary deficiency of vitamin A is associated with cardiovascular abnormalities in avian and murine systems. Retinoic acid (RA) is the active metabolite of vitamin A and whether it directly regulates mammalian blood vessel formation has not been determined and is investigated herein. We used mice rendered RA-deficient via targeted deletion of retinaldehyde dehydrogenase 2 (Raldh2(-/-)), the enzyme required to produce active RA in the embryo. Histological examination at E8.0-8.5, prior to cardiac function and systemic blood circulation, revealed that capillary plexi formed in Raldh2(-/-) yolk sacs and embryos, but were dilated, and not appropriately remodeled or patterned. Raldh2(-/-) endothelial cells exhibited significantly increased expression of phosphohistone 3 and decreased expression of p21 and p27, suggesting that RA is required to control endothelial cell cycle progression during early vascular development. Uncontrolled endothelial cell growth, in Raldh2(-/-) mutants, was associated with decreased endothelial cell maturation, disrupted vascular plexus remodeling and lack of later stages of vessel assembly, including mural cell differentiation. Maternally administrated RA restored endothelial cell cycle control and vascular patterning. Thus, these data indicate that RA plays a crucial role in mammalian vascular development; it is required to control endothelial cell proliferation and vascular remodeling during vasculogenesis.     

Retinoic acid negatively regulates p34cdc2 expression during human neuroblastoma differentiation.

p34cdc2 is a protein kinase that has an important role in controlling cell cycle progression and may regulate tumor suppressor gene activity. In this work, we show that the arrest of cell growth and induction of differentiation in a tumorigenic neuroblastoma cell line by retinoic acid (RA) is associated with a 75-fold decrease in the level of p34cdc2 protein. The RA induced decrease in p34cdc2 levels does not simply reflect the arrest of cell growth, because p34cdc2 levels are not reduced when neuroblastoma cells are growth arrested by nutrient deprivation. Furthermore, dephosphorylation of the tumor suppressor gene product RB, a substrate for the p34cdc2 kinase activity, is observed only when p34cdc2 levels are decreased in RA treated cells. These studies link regulation of cdc2 level, RB phosphorylation state, and induction of differentiation by RA and suggest that alterations in the cdc2 gene or in genes controlling its regulation contribute to tumorigenesis.