L. M. De Luca

Vitamin A deprivation results in reversible loss of hippocampal long-term synaptic plasticity

Despite its long history, the central effects of progressive depletion of vitamin A in adult mice has not been previously described. An examination of vitamin-deprived animals revealed a progressive and ultimately profound impairment of hippocampal CA1 long-term potentiation and a virtual abolishment of long-term depression. Importantly, these losses are fully reversible by dietary vitamin A replenishment in vivo or direct application of all trans-retinoic acid to acute hippocampal slices. We find retinoid responsive transgenes to be highly active in the hippocampus, and by using dissected explants, we show the hippocampus to be a site of robust synthesis of bioactive retinoids. In aggregate, these results demonstrate that vitamin A and its active derivatives function as essential competence factors for long-term synaptic plasticity within the adult brain, and suggest that key genes required for long-term potentiation and long-term depression are retinoid dependent. These data suggest a major mental consequence for the hundreds of millions of adults and children who are vitamin A deficient.

Retinoids induce tissue transglutaminase in NIH-3T3 cells.

We report that all-trans and 13-cis-retinoic acid as well as the synthetic compound CH-55 enhance tissue transglutaminase activity as they increase NIH-3T3 cell adhesiveness. The 4-hydroxyphenylretinamide (4-HPR) with low activity in inducing attachment, lectin binding and growth inhibition also fails to induce transglutaminase. Thyroxine (Thy), a compound with a response element common to RA, is inactive. The tumor promoter 12-tetradecanoyl-phorbol-13-acetate (TPA), which increases adhesiveness with different kinetics than RA, failed to enhance tranglutaminase. We conclude that retinoids with biological activity in inducing adhesion, inhibition of growth and increase of lectin binding, are also active in inducing transglutaminase activity.

β-Carotene fails to act as a tumor promoter, induces RAR expression, and prevents carcinoma formation in a two-stage model of skin carcinogenesis in male sencar mice

Clinical trials have shown a significant increase in incidence of lung cancer among smokers and asbestos workers supplemented with β-carotene, suggesting a tumor- promoting activity for this agent. We set out to test possible tumor-promoting and chemopreventive activities of dietary and topical β-carotene in the two-stage 7,12-dimethylbenz[a]anthracene-12-O-tetradecanoylphorbol 13-acetate (TPA) model of mouse skin carcinogenesis. In the first study, the effects of three levels of dietary β-carotene (6, 60, and 600 μg/g purified diet containing no other retinoid or carotenoid) were assessed over a period of 42 weeks. Carcinoma yield was reduced by ∼50% in the 600 μg/g diet group (mean 0.22 carcinomas/mouse) compared with the 6 μg/g diet group (mean 0.44 carcinomas/mouse, p = 0.003). The 60 μg/g diet group showed a pattern of inhibition similar to the 600 μg/g diet group. A protective effect (25% reduction) of β-carotene (in the 600 μg/g diet group) on papilloma formation was also found. However, the intermediate 60 μg/g diet group showed the same incidence as the low 6 μg/g diet group. This points to a lack of correlation between papilloma and carcinoma incidence, as we also found in previous work on dietary retinoids and carotenoids. The purpose of the second study was to assess whether topical β-carotene (2 μg) has tumor-promoting or chemopreventive activity in the two-stage protocol. In the absence of TPA, β-carotene had no significant tumor-promoting activity. Instead, papilloma yield induced by TPA was decreased by topical β-carotene from an average of 20 to ∼10 papillomas/mouse (p = 2.5 ×10-7). The effect of topical β-carotene persisted beyond the treatment period (Week 24) until the termination of the study at Week 42. Western blot analysis of mouse skin extracts showed that topical β-carotene upregulated retinoic acid receptor-α and -γ expression in the dorsal skin. This finding suggests that β-carotene may work as a chemopreventive agent by upregulating the expression of retinoid receptors in mouse skin. In conclusion, our data show that β-carotene prevents skin carcinoma formation, induces retinoic acid receptor expression, and fails to act as a tumor promoter in the two-stage model of skin tumorigenesis.

Altered Localization of Retinoid X Receptor α Coincides with Loss of Retinoid Responsiveness in Human Breast Cancer MDA-MB-231 Cells

ABSTRACT To understand the mechanism of retinoid resistance, we studied the subcellular localization and function of retinoid receptors in human breast cancer cell lines. Retinoid X receptor α (RXRα) localized throughout the nucleoplasm in retinoid-sensitive normal human mammary epithelial cells and in retinoid-responsive breast cancer cell line (MCF-7), whereas it was found in the splicing factor compartment (SFC) of the retinoid-resistant MDA-MB-231 breast cancer cell line and in human breast carcinoma tissue. In MDA-MB-231 cells, RXRα was not associated with active transcription site in the presence of ligand. Similarly, ligand-dependent RXR homo- or heterodimer-mediated transactivation on RXR response element or RARE showed minimal response to ligand in MDA-MB-231 cells. Infecting MDA-MB-231 cells with adenoviral RXRα induced nucleoplasmic overexpression of RXRα and resulted in apoptosis upon treatment with an RXR ligand. This suggests that nucleoplasmic RXRα restores retinoid sensitivity. Epitope-tagged RXRα and a C-terminus deletion mutant failed to localize to the SFC. Moreover, RXRα localization to the SFC was inhibited with RXRα C-terminus peptide. This peptide also induced ligand-dependent transactivation on RXRE. Therefore, the RXRα C terminus may play a role in the intranuclear localization of RXRα. Our results provide evidence that altered localization of RXRα to the SFC may be an important factor for the loss of retinoid responsiveness in MDA-MB-231 breast cancer cells.

RECENT DEVELOPMENTS IN STUDIES ON BIOLOGICAL FUNCTIONS OF VITAMIN A IN NORMAL AND TRANSFORMED TISSUES

Abstract 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 dish surface in an EDTA adhesion assay. After culturing in presence of 3.3 x 10-6 to 3.3 x 10-5M 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, retinyl-phosphate, retinoic acid, 5,6-epoxyretinoic acid and the TMMP and DACP derivatives of retinoic acid. 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 tested in this assay. Dolichol, dolichylphosphate juvenile hormone, abscisic acid, β-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 retinoid treatment at least at 6, 24, 48 and 72 hours after treatment with 3.3 x 10-5M retinoic acid, when most of the cells remain attached. Retinoic acid stimulated the incorporation of (2-3H) mannose into glycoproteins of 3T12 cells. (11, 123H 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 carried out by formalin -fixed 3T12 cells. Mild alkaline conditions 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 acid can be reduced to an alcohol, probably after metabolic modification. It is further suggested that such “retinol-like” compound would follow the same route of phosphorylation 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 to anhydroretinol in 30 minutes at 37°C. This reaction may well represent a detoxification mechanism for the transformed cell.

THE BIOSYNTHESIS OF A MANNOLIPID CONTAINING A METABOLITE OF RETINOIC ACID BY 3T12 MOUSE FIBROBLASTS

Retinol and retinoic acid (RA) increase the adhesive properties of spontaneously transformed mouse fibroblasts (BALB/c 3T12-3 cells) and the incorporation of [2-3H]mannose into cellular glycoconjugates. Therefore we searched for a mannolipid of retinoic acid similar to mannosylretinylphosphate (MRP) in these cells. Radioactively labeled RA was incorporated into a compound of chromatographic characteristics similar to those of standard MRP. This metabolite contained the same 3H:14C ratio as the precursor [11, 12-3H, 15-14C]retinoic acid, demonstrating that no decarboxylation had occurred. A doubly labeled mannolipid was obtained from cells incubated with [2-3H]mannose and [15-14C]retinoic acid. This mannolipid was readily cleaved by mild acid, yielding [3H]mannosephosphate and a compound that migrated as standard anhydroretinol on thin layer of silica gel in toluene:chloroform:methanol (4:1:1) at Rf 0.93. Standard all trans-MRP yields all-trans-anhydroretinol under these conditions. An ion pair reverse phase HPLC system was developed to further characterize the mannolipids obtained from retinol and retinoic acid in 3T12 cells. [15-3H]Retinol and [15-14C]retinoic acid were incorporated into mannolipids that cochromatographed upon HPLC with standard MRP. The mixture of the [15-3H]retinol and [15-14C]retinoic acid derived mannolipids was subjected to mild acid hydrolysis, after purification by HPLC. Nearly 100% of the compounds was hydrolyzed, yielding all-trans-[3H]anhydroretinol and a 14C]labeled product which was eluted from HPLC as a slightly more polar compound than all-trans-anhydroretinol. The retinoic acid-derived mannolipid (MXP) represented approximately 4% of the total radioactivity in the methanolic extract of 3T12 cells incubated for 20 hours in the presence of labeled retinoic acid. However, if the cells were incubated for an additional 20 hours in the absence of the radioactive precursor, MXP represented 40% of the total extracted radioactivity. These results demonstrate that 3T12 cells synthesize mannosylretinylphosphate from retinol and a mannosylretinoidphosphate (MXP) from retinoic acid. These results exclude the possibility of a reduction of retinoic acid to retinol, but suggest that a closely related compound is formed from RA and that this retinol-like compound (X) is incorporated into MXP.

Retinoids and cell adhesion

Publisher Summary This chapter summarizes the salient methodologies and pertinent data of the adhesive effects of retinoic acid and other retinoids on fibroblastic cell lines. The chapter shows the effect of different concentrations of retinoic acid on the detachment of BALB/3TI2 cells. Cells are found to adhere more strongly when cultured in the presence of retinoic acid (RA) for 3 days. The time required for cell detachment varies with the type of plastic and its preparation for tissue culture purposes. In addition to increasing the adhesiveness of fibroblasts to plastic, retinoids also enhance the ability of fibroblastic cells to attach to specific extracellular matrix components. In this investigation, the NIH 3T3 fibroblast cell system is employed for most observations, but the observed effect applies to a variety of fibroblastic cells. The retinoid also causes an increased attachment to laminin in all the nontransformed cells tested, including NIH 3T3, 3T3-Swiss, 3T6-Swiss, and BALB/3T3. In contrast, transformed fibroblasts show a higher attachment to laminin substrates as compared to their untransformed counterparts. Varied responses to RA are observed.

RETINOIDS AND CONTROL OF EPITHELIAL DIFFERENTIATION AND KERATIN BIOSYNTHESIS IN HAMSTER TRACHEA

Publisher Summary This chapter describes the methodologies utilized in the assessment of the morphological and biochemical alterations that occur during vitamin A deficiency in the hamster tracheal epithelium. The tracheal epithelium is of the pseudostratified type—that is, all its cells are in contact with the basement membrane. The epithelial tracheal surface fulfills its function of clearing foreign particles by virtue of the movement of the cilia. These, in turn, are aided in their motion by the presence of the mucus secreted by the goblet cells. Mechanical or chemical injury causes focal replacement of the mucociliary epithelium by a squamous metaplastic phenotype. Eventual reestablishment of the mucociliary epithelium after injury takes place gradually. In animals lacking vitamin A, a similar process of loss of the mucociliary epithelium and squamoid metaplasia is also observed. Retinoids at very low concentrations permit the regeneration of the normal mucociliary phenotype in vivo as well as in organ and cell culture. The morphological changes arising from deficiency of vitamin A are usually assessed at the midpoint of the tracheas. This is in part because the larynx is normally squamoid and keratinized.