Protein and ligand adaptation in a retinoic acid binding protein.

A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all-trans and 9-cis isomers, the structures of all-trans retinoic acid-ERABP and 9-cis retinoic acid ERABP were determined. Our results indicate that the all-trans isomer of retinoic acid adopts an 8-cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB-ERABP is also reported herein. To accommodate this all-trans analog, which cannot readily adopt a cis-like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo-proteins.     

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RAR alpha, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.     

Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors

Vitamin A deficiency has been known for a long time to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Increasing evidence suggests that retinoic acids derived from vitamin A are involved in the functional regulation of the immune system. Of the two groups of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) all-trans and 9-cis retinoic acids are high affinity ligands for RARs and 9-cis retinoic acid additionally binds to RXRs. In cells, at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid by unknown mechanisms. Apoptosis plays a major role in shaping the T cell repertoire and one way in which retinoids may affect immune functions is to influence the various apoptosis pathways. Indeed, it has been shown that retinoic acids can induce apoptosis, increase the rate of dexamethasone-induced death and inhibit activation-induced death of thymocytes and T lymphocytes. Therefore, retinoids together with glucocorticoids may be involved in regulating positive and negative selection of T lymphocytes. Here we demonstrate that retinoids can induce apoptosis of T cells through the stimulation of RARgamma. Specific stimulation of RARalpha, on the other hand, prevents both RARgamma-dependent and TCR-mediated cell death. In all these functions 9-cis retinoic acid proved to be more effective than all-trans retinoic acid suggesting the involvement of RXRs. Based on these results a possible mechanism through which costimulation of RARs and RXRs might affect spontaneous and activation-induced death of T lymphocytes is proposed.     

Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes.

It has been shown that all-trans retinoic acid induces prematurely hepatic glucokinase mRNA in ten days-old neonatal rat hepatocytes, however, this effect could be related to the capacity of the retinoid to promote a more differentiated state of the hepatocyte. In this report we demonstrate that physiological concentrations of all-trans retinoic acid stimulate glucokinase activity in both mature fully differentiated hepatocytes and at the onset of the induction of the enzyme in 15 to 17 days-old neonatal hepatocytes. The effects produced by the retinoid were similar both in magnitude and in time, to those elicited by insulin, a well-known stimulator of hepatic glucokinase expression. No additive effect was observed when insulin and retinoic acid were tested together. Using the branched DNA assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of about 70% at 3 and 24 h after the treatment with 10(-6) M all-trans retinoic acid, in both neonatal and adult hepatocytes. These data show that retinoic acid exerts a stimulatory effect on hepatic glucokinase independent of the hepatocyte stage of maturity and suggest a physiological role of retinoic acid on glucose metabolism. The action of retinoic acid on hepatic glucokinase might explain previous observations on the relationship between vitamin A status and liver glycogen synthesis. These findings may serve as basis for further investigations on the biological functions of retinoic acid derivatives on hepatic glucose metabolism.     

Effect of retinoic acid on two glycosyltransferase activities in C6 cultured glioma cells

1. Activity of two glycosyltransferases was studied in retinoic acid-treated C6 cultured glioma cells. 2. The beta-galactoside alpha 2,3-sialyltransferase transferring N-acetylneuramin onto the O-glycans residues of glycoproteins was activated up to twice after chronic treatment (from 24 to 96 hr) with all-trans retinoic acid. 3. No effect was observed for shorter treatments. 4. On the opposite, the N-glycan galactosyltransferase activity remained unchanged whatever the length of retinoic acid treatment was. 5. The activatory effect was not dependent on isomery, as all-trans and 13-cis retinoic acid isomers were both activators of the C6 glioma cell sialyltransferase. 6. Measurement of adhesion of retinoic acid-treated cells using labelled plasma membranes showed an enhancement of adhesion in correlation with enhancement of sialyltransferase activity.     

Conditioned media from solid tumor cell lines treated with retinoic acids both decreases and increases proliferation of capillary endothelial cells.

These studies investigated the effects of retinoic acids on endothelial cell proliferation. Three human neoplastic cell lines, U-373 MG glioblastoma, DU-145 prostate carcinoma, and TCCSUP bladder transitional cell carcinoma, were treated with all-trans, 9-cis, or 13-cis retinoic acids at 0.0001 to 10 microM. Hypoxia was used to ensure the expression of the angiogenic phenotype. Conditioned media (CM) were prepared by hypoxic culturing of the tumor cells with retinoic acids for 24 hours. Then CM were transferred to bovine capillary endothelial cells for 48 hours of normoxic culturing, counted and compared to controls. CM from U-373 MG and DU-145 cells, but not TCCSUP cells, treated with all-trans or 9-cis retinoic acids at several concentrations below 1 microM, caused significant (P<0.05) increases in endothelial cell proliferation of between 13 to 18%. Both nonconditioned and conditioned media, for retinoic acid concentrations above 1 microM, inhibited endothelial cell proliferation. All CM for 13-cis retinoic acid decreased endothelial cell proliferation. These results show that the cytotoxicity of retinoic acids and the growth promoting/inhibiting ability of the conditioned media is retinoic acid isoform, time, concentration, and cell type dependent. Most importantly, the conditioned media from tumor cells treated with low concentrations of all-trans or 9-cis retinoic acids significantly increased endothelial cell proliferation.     

Teratogenicity of all-trans retinoic acid during early embryonic development in the cynomolgus monkey (Macaca fascicularis).

The embryotoxic and teratogenic potential of all-trans retinoic acid was assessed following exposure prior to and during early organogenesis in the cynomolgus monkey (Macaca fascicularis). Sixteen pregnant females were orally administered all-trans retinoic acid (Tretinoin, Hoffmann-La Roche) once daily from GD 10-20 and twice daily from GD 21-24 at three different dosages, 5 (n = 9), 10 (n = 6) and 20 mg/kg (n = 1). Adverse clinical signs resembling hypervitaminosis A were observed in one animal at 5 mg/kg, in three animals at 10 mg/kg, and in the animal treated with 20 mg/kg all-trans retinoic acid. Maternal weight loss was observed in the 10- and 20-mg/kg groups. A dose-dependent increase in embryolethality was observed, with 22% (2/9), 50% (3/6), and 100% (1/1) occurring at 5, 10, and 20 mg/kg, respectively. The majority of embryonic deaths occurred between GD 16 and 20; the incidence of these early losses was higher than in historical and concurrent controls. No malformations, but a single growth-retarded fetus, was observed in the 5-mg/kg group. Craniofacial malformations, consisting of external ear defects, mandibular hypoplasia, cleft palate, and temporal bone abnormalities, were seen in three viable fetuses in the 10-mg/kg group. Skeletal variations were common to the majority (70%, 7/10) of viable fetuses in both dose groups and were increased relative to historical controls (32%, 25/77). Unlike previous studies with 13-cis-retinoic acid during the pre- and early organogenic stages of development (Hummler et al., Teratology 42:263-272, 1990), no thymic hypo- or aplasia or heart anomalies were observed, which may be attributable to the slightly longer 13-cis retinoic acid treatment period, i.e., GD 10-27. However, external ear and temporal bone defects were common to both all-trans and 13-cis retinoic acid. The similarity observed in the malformation syndrome induced by both all-trans and 13-cis retinoic acid in the cynomolgus monkey and 13-cis retinoic acid embryopathy in humans supports this macaque species as a model for further developmental toxicity studies of vitamin A-related compounds.     

Synergistic effect of vitamin D derivatives and retinoids on C2C12 skeletal muscle cells

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.     

Interaction between the aryl hydrocarbon receptor and retinoic acid pathways increases matrix metalloproteinase-1 expression in keratinocytes

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of pathological lesions in humans via activation of the aryl hydrocarbon receptor (AhR) pathway. It has become apparent that this pathway interacts with a variety of signaling pathways that are believed to be involved in mediating TCDD/AhR biological effects. Our hypothesis is that TCDD mediates these pathological lesions by directly altering the expression of genes involved in matrix deposition and remodeling and that the retinoic acid signaling pathway is involved in modulating TCDD-induced effects. Therefore, we examined the effect of TCDD and all-trans retinoic acid (atRA) on the expression of matrix metalloproteinase-1 (MMP-1, interstitial collagenase), one of the proteolytic enzymes that degrade type I collagen, in normal human keratinocytes. The data show that TCDD exposure results in increased MMP-1 expression in keratinocytes that is further enhanced by co-treatment with all-trans retinoic acid. TCDD-induced expression of MMP-1 appears to be mediated through two AP-1 elements in the proximal promoter of the MMP-1 gene. However, retinoic acid-mediated induction of keratinocyte MMP-1 is a result of both promoter activation and increased mRNA stability. These findings are the first to demonstrate TCDD-induced expression of MMP-1 and to demonstrate interactions between the TCDD/AhR and retinoic acid pathways on MMP-1 expression.     

Retinoic acid exacerbates experimental radiation nephropathy

Studies have shown that angiotensin-converting enzyme inhibitors and an angiotensin II receptor blocker can delay, but cannot reverse, the progression of experimentally induced radiation nephropathy. In an effort to find a method for reversing injury, three agents were tested in a rat model of radiation nephropathy. Pirfenidone (a phenyl-pyridone antifibrotic) and thiaproline (an inhibitor of collagen deposition) were not capable of retarding the development of radiation nephropathy. However, all-trans retinoic acid (an anti-inflammatory agent) exacerbated radiation nephropathy. We speculated that the detrimental effects of retinoic acid might be the result of stimulation of renal cell proliferation. However, retinoic acid had no effect on tubular or glomerular cell proliferation in normal animals and did not enhance radiation-induced proliferation. A recent report that retinoic acids inhibit nitric oxide production suggested an alternative mechanism, since inhibition of production of nitric oxide is known to exacerbate radiation nephropathy. Experiments demonstrated that retinoic acid exacerbated the radiation-induced drop in renal production of nitric oxide, suggesting that the detrimental effect of all-trans retinoic acid might be explained by inhibition of renal nitric oxide activity. Particularly in view of the recent clinical report of enhancement of radiation nephropathy by retinoic acid in patients receiving bone marrow transplantation, the combination of retinoic acid and renal irradiation should be carried out with great caution.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Heterodimeric BMP-2/7 Antagonizes the Inhibition of All-Trans Retinoic Acid and Promotes the Osteoblastogenesis

Objectives

Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration.

Materials and Methods

We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points.

Results

All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28^th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7).

Conclusions

Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism.     

Retinoic acid formation from retinol in the human gastric mucosa: role of class IV alcohol dehydrogenase and its relevance to morphological changes

Alcohol dehydrogenase (ADH) participates in the formation of retinoic acid from retinol in various organs including the gastric mucosa. However, its clinical significance still remains to be clarified. In this study, we identified the ADH isoforms responsible for the retinoic acid formation among various ADH isoforms and examined associations among the ADH activities, the retinoic acid formation level, and morphological changes in the human gastric mucosa. Human gastric samples were endoscopically obtained from 67 male subjects. Morphological changes were assessed by the Sydney system and activities of class I, III, and IV ADH isoforms were determined in each specimen. In 26 cases, levels of all-trans retinoic acid (ATRA) formation from all-trans retinol were examined. Among activities of the three ADH isoforms, class IV ADH activity was solely associated with the ATRA formation level. This association was found even when subjects' age and Helicobacter pylori infection status were adjusted. As the degrees of inflammation, atrophy, and intestinal metaplasia increased, the class IV ADH activity as well as the potential for the ATRA formation decreased. Class IV ADH is a major enzyme in the retinoic acid supply in the human gastric mucosa, and the reduction of its activity was associated with decreasing retinoic acid supply and progression of inflammation, atrophy, and intestinal metaplasia in the gastric mucosa. In that retinoic acid is a key molecule for maintaining normal morphology, the reduction of class IV ADH activity may be involved in the pathogenesis of these morphological changes in the human gastric mucosa.