Retinoid metabolism and mode of action.

Vitamin A and its derivaties (retinoids) are necessary for the maintenance of normal phenotypic expression. An attempt at understanding the biochemical role of vitamin A had led to the demonstration of a new pathway for retinol. In this pathway, vitamin A is phosphorylated to retinylphosphate (RP), which is then glycosylated to retinylphosphatemannose (MRP). These two derivatives have been found in a variety of tissues in vivo and in vitro and appear to be ubiquitous components of cellular membranes. The suggestion has been made that MRP may mediate specific cellular interactions by functioning as a lipid intermediate in the biosynthesis of specific glycoconjugates. A study on spontaneously-transformed mouse fibroblasts (Balb/c 3T12-3 cells) has shown that retinoids are active in increasing the adhesive properties of these cells as measured in an EDTA-mediated detachment assay. Various retinoids were tested for their activity in the adhesion test, and this activity was found to correlate well with their biological activity in maintaining the expression of normal epithelial differentiation in other systems. Retinoic acid, 5,6-epoxyretinol, and 5,6-epoxyretinoic acid were the most active compounds. Retinoids without biological activity in other systems were also inactive in inducihg adhesive properties of 3T12-3 cells. Among these were the synthetic derivatives of retinol, anhydroretinol, and 4,5-monoeneperhydroretinol, and the phenyl derivative of retinoic acid. Beta-Ionone, abscisic acid, and juvenile hormone, which are devoid of vitamin A activity in other systems, were also inactive in this system. Retinoid-induced changes in cell surface proteins were investigated but no difference in 125I-fibronectin (MW 220,000) was detectable between retinoid-treated and untreated cells. However, these cells synthesized retinylphosphatemannose and the incorporation of 2-3H-mannose into a specific glycoprotein (gp 180) was found to be enhanced specifically by retinoid treatment. Investigations of the involvement of gp 180 in adhesion are in progress

Retinoid-induced adhesion in cultured, transformed mouse fibro- blasts.

Cultured, spontaneously transformed mouse fibroblasts (Balb/3T12-3 cells) were readily detached from the dish surface in an EDTA-mediated detachment assay. Retinoic acid-treated cells displayed increased adhesion to the culture dish surface. The effect of retinoic acid on the adhesion of Balb/3T12-3 cells was dose-dependent in the range of 0.05-5 μg/ml (0.17-17 μM) in an assay performed on cells cultured for 3 days in the presence of the retinoid. The earliest effect on adhesion was detected at 2 days of culture in the presence of 17 μM retinoic acid. The increase in adhesion displayed by retinoic acid-treated cells was rapidly lost upon removal of the retinoid from the culture medium. Synthetic retinoids were tested for their activity in inducing adhesion of cultured Balb/3T12-3 cells. Retinol, retinoic acid, and their 5,6-epoxy derivatives were the most active, showing activity at 1 μg/ml. 13-cis-Retinoic acid and the dimethylacetylcyclopentenyl and trimethylmethoxyphenyl derivatives were active at 10 μg/ml. However, active derivatives of retinoic acid invariably lost their activity upon chemical esterification or amide formation. Retinoids without biologic activity in other systems were also inactive in inducing adhesion. Among these were the synthetic derivatives of retinol, anhydroretinol and perhydromonoeneretinol, and the phenyl derivative of retinoic acid. β-Ionone, abscisic acid, and juvenile hormone were also inactive. Results showed that this adhesion assay may be used as an additional test for the biologic activity of retinoids containing a free carboxylic or carbinolic function. The phenomenon of induced adhesion may also aid in the study of the metabolic function of vitamin A

Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol

Spontaneously transformed mouse fibroblasts (Balblc 3T 12-3 cells) displayed an increased adhesion when cultured in the presence of loF6 M all-trans retinol and acquired morphological characteristics of the normal phenotype. Thus it was of interest to investigate the metabolism of [ 15-14C]retinol in this system. Within 24 hours of culture, approximately 4.25% of the [14C]retinol was taken up by the cells. The hydrocarbon [14C]anhydroretinol was a major metabolic product and was identified by gas-liquid chromatography and by its typical ultraviolet absorption spectrum with maxima at 386, 364, and 346 nm. At 24 and 40 hours anhydroretinol represented 27% and 55%, respectively, of the total nonpolar metabolites or approximately 16% and 30% of the total radioactive products. Formalin-fixed fibroblasts or cultured intestinal mucosal cells did not convert retinol into anhydroretinol. A more polar product with a UV absorption maximum at 310 nm was also found. The time course of the synthesis of this product by 3T12 cells suggested a precursor-product relationship with anhydroretinol. A microsomal preparation from 3T 12 cells was also active in synthesizing [14C]anhydroretinol and [14C]metabolite- 3 10 from [14C]retinol. Moreover incubation of metabolite-3 10 with the 3T12 microsomes yielded anhydroretinol(40% conversion in 30 minutes), suggesting that metabolite-310 is an intermediate in the synthesis of anhydroretinol by these cells. Anhydroretinol appears to be an end product of the metabolism of retinol in 3T12-3 cells, as suggested by the finding that over 90% of [14C]anhydroretinoli ncubated for 30 hours with 3T12-3 cells was recovered unaltered, without the formation of detectable retroretinol, retinol, or retinoic acid.- Bhat, P. V., L. M. De Luca, S. Adamo, I. Akalovsky, C. S. Silverman-Jones, and G. L. Peck. Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balbk 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol. J . Lipid Re\. 1979. 20: 357-362

Retinoid metabolism and mode of action

Vitamin A and its derivatives (retinoids) are necessary for the maintenance of normal phenotypic expression. An attempt at understanding the biochemical role of vitamin A has led to the demonstration of a new pathway for retinol. In this pathway, vitamin A is phosphorylated to retinylphosphate (RP), which is then glycosylated to retinylphosphatemannose (MRP). These two derivatives have been found in a variety of tissues in vivo and in vitro and appear to be ubiquitous components of cellular membranes. The suggestion has been made that MRP may mediate specific cellular interactions by functioning as a lipid intermediate in the biosynthesis of specific glycoconjugates. A study on spontaneously-transformed mouse fibroblasts (Balb/c3T12-3 cells) has shown that retinoids are active in increasing the adhesive properties of these cells as measured in an EDTA-mediated detachment assay. Various retinoids were tested for their activity in the adhesion test, and this activity was found to correlate well with their biological activity in maintaining the expression of normal epithelial differentiation in other systems. Retinoic acid, 5,6-epoxyretinol, and 5,6-epoxyretinoic acid were the most active compounds. Retinoids without biological activity in other systems were also inactive in inducing adhesive properties of 3T12-3 cells. Among these were the synthetic derivatives of retinol, anhydroretinol, and 4,5-monoeneperhydroretinol, and the phenyl derivative of retinoic acid. β-Ionine, abscisic acid, and juvenile hormone, which are devoid of vitamin A activity in other systems, were also inactive in this system. Retinoid-induced changes in cell surface proteins were investigated but no difference in 125I-fibronectin (MW 220,000) was detectable between retinoid-treated and untreated cells. However, these cells synthesized retinylphosphatemannose and the incorporation of 2-3H-mannose into a specific glycoprotein (gp 180) was found to be enhanced specifically by retinoid treatment. Investigations of the involvement of gp 180 in adhesion are in progress

Recent developments in studies on biological functions of vitamin A in normal and transformed tissues

A biochemical pathway of phosphorylation and glycosylation of vitamin A has recently been found in hepatic, intestinal and epidermal tissues. More recent work suggests that mannosylretinylphosphate functions as a donor of mannose to membrane glycoconjugates. These reactions might ultimately explain the effects of vitamin A deficiency and some of the effects of excess vitamin A on biological systems. Studies of the effect of retinoids on cellular in vitro systems showed an increase in the adhesive properties of spontaneously-transformed mouse fibroblasts in culture (Balb/c 3T12-3 cells). These cells are usually detached from the culture gish surface ig an EDTA adhesion assay. After culturing in presence of 3.3 x 10- to 3.3 x 10- M retinol or retinoic acid the cells are no longer lifted from the plate and their morphology and adhesion resemble those of normal fibroblasts. This phenomenon of increased adhesion is observed as early as two days after exposure to the retinoid and it is readily reversible upon culturing in medium without exogenous retinoid. A variety of retinoids was tested in the adhesion assay. The most active compounds were retinol, retinylphosphate, retinoic acid, 5,6-epoxyretinoic acid and the TMMP and DACP derivatives of retinoic acido All these compounds possess biological activity in other systems. Anhydroretinol, perhydromonoeneretinol, the" phenyl derivative of retinoic acid, which do not have biological activity in other systems, did not increase adhesion of 3T12 cells. Other polyprenoid compounds without vitamin A activity were also test ed in this assay. Dolichol, dolichylphosphate juvenile hormone, abscisic aCid, s-ionone, dibutyryl cyclic adenosine monophosphate and sodium butyrate did not induce adhesion. The mechanism by which retinol and retinoic acid increase the adhesive properties of 3T12 cells was investigated. Cyclic adenosine monophosphate and guanosine monophosphate levels were not significantly altered by retino~d treatment at least at 6, 24, 48 and 72 hours after treatment with 3.3 x 10- M retinoic acid, when most30f the cells remain attached. Retinoic acid stimulated the inc~rporation of (2- H) mannose into glycoproteins of 3T12 cells. (11, 12 H and carboxyl-14C)Retinoic acid was incorporated into a compound (Metabolite I) which had chromatographic properties of a glycosylretinylphosphate. The synthesis of this compound was time-dependent and was not carri ed out by formalin -fixed 3T12 cells. Mild alkaline conditiQns which release anhydroretinol from retinylphosphate, also cleaved Metabolite I to yield a product with the polarity of a hydrocarbon, but slightly more polar than anhydroretinol. It is suggested that retinoic atid can be reduced to an alcohol, probably after metabolic modification. It is further suggested that such "reti nol-l i ke" compound woul d fo" ow the same route of phosphoryl at ion and glycosylation as shown for retinol in other systems. Microsomes from 3T12 cells were active as the intact cells in synthesizing mannosylretinylphosphate and dolichyl mannosylphosphate. Exogenous retinylphosphate specifically stimulated the synthesis of mannosylretinylphosphate. Thus it appears that vitamin A is involved in glycosyl transfer reactions in the 3T12 system, as well as in normal membranes. It remains to be established whether the observed increased adhesion is the result of such involvement. A novel reaction for retinol was found in 3T12 cells. Up to 55% of exogenously supplied retinol was converted to the hydrocarbon anhydroretinol in 48 hours. The same reaction was also carried out by microsomes from 3T12 cells, which converted 7% of retinol toanhydroretinol in 30 minutes at 370C. This reaction may well represent a detoxification mechanism for the transformed cell