Cellular retinoic acid binding protein: detection and quantitation in regenerating axolotl limbs

The concentrations of apo (unoccupied), holo (occupied), and total cellular retinoic acid binding protein (CRABP) were measured at various stages of axolotl limb regeneration. The ratio of apo-CRABP to holo-CRABP declined with advancing regenerate stage until the CRABP was all in the holo form. The increase in holo-CRABP is correlated with a stage-dependent shift in the effect of exogenous retinoic acid on regenerate pattern, from pattern duplication to inhibition of regeneration. The data suggest, though they do not prove, that these different morphological effects could be due to a shift from a CRABP-dependent to a CRABP-independent mechanism of exogenous retinoic acid (RA) action that is related to stage-specific variations in endogenous RA levels.     

The relationship among retinoid structure, affinity for retinoic acid-binding protein, and ability to respecify pattern in the regenerating axolotl limb

To further our understanding of the action of retinoids on the respecification of pattern in the regenerating axolotl limb we have studied the relative potencies of a range of synthetic and natural retinoids administered locally to the blastema. Alterations in the polar end group of the retinoic acid (RA) molecule to produce esters, the alcohol, or the aldehyde abolish the ability of the molecule to respecify pattern. On the other hand, alterations of the ring or side chain to produce the synthetic retinoids arotinoid and TTNPB considerably increases the potency of the molecule to respecify pattern--TTNPB is at least 100X more potent than retinoic acid. To examine the role of cellular retinoic acid-binding protein (CRABP) in the respecification process we determined the relative binding affinities of these retinoids for CRABP. These data correlated well with the respecification series: retinoids which showed no affinity for CRABP did not respecify pattern and those which did show affinity for CRABP did respecify pattern. Furthermore the most potent retinoid, TTNPB, has a higher affinity for CRABP than RA itself. This suggests that CRABP may be playing an important role in the action of RA on pattern formation in the regenerating limb.     

The respecification of limb pattern by new synthetic retinoids and their interaction with cellular retinoic acid-binding protein

We describe here experiments to examine the role of cellular retinoic-acid-binding protein (CRABP) during the induction of limb duplication in the chick limb bud and regenerating axolotl limb by retinoids. A newly synthesised class of retinoic acid analogues have been used because among them, some have been specifically designed with the property of binding to the retinoic acid receptors, but not to CRABP. We can thus test whether binding to CRABP is an obligatory step during limb respecification. The binding of four of these compounds to chick limb bud and axolotl CRABP was tested in sucrose density gradient assays and then their potencies at inducing limb duplications tested. Two of the four compounds do not bind to limb CRABP and yet are able to induce limb duplications, suggesting that an interaction with CRABP is not an obligatory step in the process. However, the two compounds which do bind to CRABP are more potent than the two which do not, suggesting that an interaction with CRABP may, nevertheless, increase the potency of a retinoid.     

Retinoic acid-binding protein in the axolotl: distribution in mature tissues and time of appearance during limb regeneration

Analysis of cytoplasmic protein preparations from axolotl tissues revealed the presence of a cytoplasmic retinoic acid-binding protein (CRABP), of approximate molecular weight 17K. This protein was found to be present at various concentrations in skin, muscle, and limb tissue preparations, but not in liver and serum preparations. The distribution and molecular weight of this protein agrees with that reported in mammalian studies. The level of CRABP in cone stage blastemas was found to be significantly higher than that found in nonregenerating whole limb preparations. The level falls gradually, to approach normal, towards the completion of regeneration. Such an increase, at the start of regeneration, was not altered by 4 days pretreatment with 36 mg/liter all-trans-retinoic acid, a sufficient dose to produce pattern effects. Competition experiments confirmed that the all-trans and 13-cis isomers of retinoic acid bind to CRABP with similar high efficiencies, and that the arotinoid, Ro 13-6298, exhibits only a fraction of this binding activity. Retinol, retinol palmitate, and retinol acetate were unable to compete with [3H]retinoic acid for binding to CRABP. The results presented here are discussed in terms of their possible value to understanding pattern specification in the regenerating urodele limb.     

Use of retinoids to analyze the cellular basis of positional memory in regenerating amphibian limbs

Cells of the amphibian limb regeneration blastema inherit memories of their level of origin (positional memory) along the limb axes. These memories serve as boundaries of what is to be regenerated, thus preventing regeneration of any but the missing structures. Because of its importance in determining the boundaries of regenerate pattern, it is essential to understand the cellular and molecular basis of positional memory. One approach to this problem is to look for position-related differences in a cell or molecular property along a limb axis and then show, using an agent that modifies regenerate pattern, that the cell or molecular property and the pattern are coordinately modified. We have done this using retinoic acid (RA) as a pattern-modifying agent and an in vivo assay that detects position-related differences in a cell recognition-affinity property along the proximodistal (PD) axis of the regenerating axolotl limb. RA proximalizes positional memory in the PD axis, posteriorizes it in the anteroposterior axis, and ventralizes it in the dorsoventral axis. The level-specific PD cell recognition-affinity property is proximalized by RA, indicating that this property and positional memory are causally related. The effects of RA on positional memory may be mediated through a cellular RA-binding protein (CRABP), since the concentration of unbound (apo) CRABP molecules is highest during early stages of regeneration when the proximalizing effects of RA are greatest.     

Retinoid antagonists inhibit normal patterning during limb regeneration in the axolotl, Ambystoma mexicanum

Retinoic acid (RA) has been detected in the regenerating limb of the axolotl, and exogenous RA can proximalize, posteriorize, and ventralize blastemal cells. Thus, RA may be an endogenous regulatory factor during limb regeneration. We have investigated whether endogenous retinoids are essential for patterning during axolotl (Ambystoma mexicanum) limb regeneration by using retinoid antagonists that bind to specific RAR (retinoic acid receptor) or RXR (retinoid X receptor) retinoid receptor subtypes. Retinoid antagonists (Ro41-5253, Ro61-8431, LE135, and LE540) were administered to regenerating limbs using implanted silastin blocks loaded with each antagonist. The skeletal pattern of regenerated limbs treated with Ro41-5253 or Ro61-8431 differed only slightly from control limbs. Treatment with LE135 inhibited limb regeneration, while treatment with LE540 allowed relatively normal limb regeneration. When LE135 and LE540 were implanted together, regeneration was not completely inhibited and a hand-like process regenerated. These results demonstrate that interfering with retinoid receptors can modify pattern in the regenerating limb indicating that endogenous retinoids are important during patterning of the regenerating limb.     

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

Expression of cellular retinoic acid-binding protein I and II (CRABP I and II) in embryonic mouse hearts treated with retinoic acid

Cellular retinoic acid binding proteins are considered to be involved in retinoic acid (RA) signaling pathways. Our aim was to compare the expression and localization of cellular retinoic acid binding proteins I and II (CRABP I and II) in embryonic mouse hearts during normal development and after a single teratogenic dose of RA. Techniques such as real-time PCR, RT-PCR, Western blots and immunostaining were employed to examine hearts from embryos at 9-17 dpc. RA treatment at 8.5dpc affects production of CRABP I and II in the heart in the 48-h period. Changes in expression of mRNA for retinaldehyde dehydrogenase II (Raldh2), Crabp1 and Crabp2 genes also occur within the same time window (i.e. 10-11dpc) after RA treatment. In the embryonic control heart these proteins are localized in groups of cells within the outflow tract (OT), and the atrioventricular endocardial cushions. A gradient of labeling is observed with CRABP II but not for CRABP I along the myocardium of the looped heart at 11 dpc; this gradient is abolished in hearts treated with RA, whereas an increase of RALDH2 staining has been observed at 10 dpc in RA-treated hearts. Some populations of endocardial endothelial cells were intensively stained with anti-CRABP II whereas CRABP I was negative in these structures. These results suggest that CRABP I and II are independently regulated during heart development, playing different roles in RA signaling, essential for early remodeling of the heart tube and alignment of the great arteries to their respective ventricles.     

The molecular cloning and expression of two CRABP cDNAs from human skin

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.     

Retinoic acid proximalizes level-specific properties responsible for intercalary regeneration in axolotl limbs

The objective of this study was to determine whether retinoic acid (RA) coordinately proximalizes positional memory and the cellular recognition system that detects pattern discontinuity in regenerating amphibian limbs. The strategy was to test the capacity of RA-treated blastemas to evoke intercalary regeneration when grafted to an amputation level proximal to their level of origin. Control wrist and ankle, or elbow and knee blastemas treated with the retinoid solvent, dimethylsulphoxide, evoked intercalary regeneration as effectively as untreated blastemas, when grafted to the midstylopodial amputation surface of host limbs. RA-treated wrist and ankle or elbow and knee blastemas were proximalized and formed complete limbs that were at an angle to, or continuous with, the midstylopodium of the host limb. No intercalary regeneration, from either graft or host, was observed in these cases. The results indicate that the cellular mechanism that recognizes disparities between non-neighbouring cells and initiates intercalary regeneration is coordinately proximalized with positional memory. Thus the recognition mechanism and positional memory are directly related. Intercalary regeneration and corrective displacement (affinophoresis), both of which restore a pattern of normal cell neighbours by different means in regenerating axolotl limbs, appear to use the same mechanism to recognize pattern discontinuity.     

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.     

Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.