Many of the effects of retinoic acid on development occur through the ability of retinoic acid to regulate gene expression in various specific cell types. Retinoic acid binds to proteins in the steroid receptor family (Figure 1), and it is this RA receptor complex which mediates gene expression.
Figure 1: Structures of vitamin A and its derivatives (from Means and Gudas, 1995). Clockwise, beginning with retional (Vitamin A, in the center): Retinyl ester (RE) (STORAGE), all-trans-retinoic acid (DIFFERENTIATION), 0-cis retinoic acid, retinaldehyde (VISION), Anhydroretinol (AR) (GROWTH INHIBITION), 14-Hydroxy-Retro-Retinol (14-HRR) (GROWTH SUPPORT)
These proteins have been grouped into two subgroups, the retinoic response elements (RARs) and the retinoid "X" receptors (RXRs). The RARs a, b, and g bind to all-trans RA and its isomer 9-cis RA. The RXRs a, b, and g bind 9-cis RA, but not to all-trans RA with high affinity (Langston and Gudas, 1994). While most teratogenicity studies have focused on all trans-retinoic acid, its isomers 9-cis-retinoic acid and 13-cis-retinoic acid also may be somewhat teratogenic (Means and Gudas, 1995). Amino acid sequence comparisons have shown us that conservation of a given RAR or RXR type is greater between the species than is found between the three RAR or RXR types within a species. This conservation within a species has led to the conclusion that each RAR and RXR type has a unique function. They also have been found to synergize and activate several RA-responsive promoters (Chambon, 1993). One isoform of RAR a and one isoform of RAR g have been found to be essential for the transduction of the retinoid signal in embryos and adults. Mutation of these isoforms are lethal. Knocking out RAR a and RAR g in the limb bud, however, has not been found to effect limb morphogenesis, though its regulation is thought to be controlled by a morphogenetic gradient of RA (Chambon, 1993). Chambon (1993) mentions that isoforms which have been knocked out so far have no known phenotype. Mouse embryos which were null homozygous for RAR a and RAR g, though born without any abnormal phenotype, were either selectively eaten by the mother or died 1-2 weeks after birth. Their development was slowed and they became emaciated and lethargic, although no apparent malformations or lesion could be found (Chambon, 1993). Since growth continues after birth, the affects of these null mutations may not be affecting growth of limbs, nervous system, etc., when the embryo is initially developing, but it may be affecting the finishing touches of embryonic growth after birth when higher concentration levels may be required to regulate genes expressed or repressed at the end of embryonic development.
Another interesting finding is the high degree of functional redundancy between RAR isoforms. This is not expected because of the high conservation of all RARs and their isoforms and the specificity of their pattern of expression. The presence of duplications has likely occurred because the RARs came from a common ancestral gene, and thus have similar structures that allow it to perform similar functions. Each retinoic acid receptor is thought to have at least one unique function to "justify (its) striking conservation across vertebrates from fish to (hu)man" (Chambon, 1993).
The RAR and RXR genes have been found to activate or repress the expression of other genes. The RAR genes contain two promoters, which allow two different types of transcripts to be made. RARs can bind to RA response elements (RAREs) and directly activate gene expression Secondary changes in development not involving the binding of an RA-receptor complex to RARE, such as the expression of genes encoding for glycoproteins involved in the extracellular matrix, the tissue plasminogen activator, intermediate growth factors, can also occur through retinoic acid. While retinoic acid can regulate gene expression positively through the binding of the RA-receptor complex with RAREs, negative regulation is also mediated through an AP-1 binding site (Gudas, 1994). The RA-receptor complex prevents the activation of the genes by the AP-1 transcription factors. All cells examined so far have been found to express at least one of the RAR and RXR genes.