Retinol oxidation to retinoic acid in human thyroid glandular cells

Abstract

Retinoic acid is regarded as the retinol metabolite that controls proliferation and differentiation of epithelial cells. In the present study, we investigated the potential role of xanthine dehydrogenase (XDH) in retinoic acid biosynthesis in human thyroid glandular cells (HTGC). In particular, we observed that cellular retinoids binding proteins (CRBPs) are also implicated in the biosynthetic pathway leading to retinoic acid formation in primary cultures of HTGC, as we have already reported for human mammary epithelial cells (HMEC). After partial protein purification, the enzyme responsible for retinoic acid biosynthesis was identified and quantified as XDH by immunoassay, by its ability to oxidize xanthine to uric acid and its sensitivity to the inhibitory effect of oxypurinol. The evidence of XDH-driven formation of retinoic acid in HTGC cultures further corroborates the potential role of XDH in retinoic acid biosynthesis in the epithelia.     

Retinoic acid modulates dome formation by MDCK cells in defined medium

Retinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K-1). After 4-5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7 days with 3 x 10(-7) M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 x 10(-3) microns 2 in area, whereas in control medium the majority of the domes were between 0 and 9 x 10(-3) microns 2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K-1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1-independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K-1 lacking PGE1. This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1-independent MDCK cells. Dibutyryl cAMP-resistant MDCK cells, which normally do not form domes in medium K-1, were also studied. Remarkably, the dibutyryl cAMP-resistant MDCK cells were observed to form domes at a significant frequency when medium K-1 was supplemented with retinoic acid. However in medium K-1 lacking PGE1, an effect of retinoic acid on dome formation by dibutyryl cAMP-resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP-resistant MDCK cells to form domes in medium K-1 has previously been attributed to their decreased cAMP-dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP-dependent protein kinase or activation of a separate pathway.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

Effect of retinoic acid on proliferation and differentiation of cultured chondrocytes in terminal differentiation

We obtained terminally differentiated chondrocytes in monolayer culture from chick embryonal growth plates, and examined the effect of retinoic acid on these cells. The cells treated with retinoic acid ceased type X collagen synthesis and showed decreased calcium incorporation into cell layers. Retinoic acid tended to stimulate proliferation of the cultured chondrocytes. It also increased DNA accumulation dose-dependently in the range from 1 nM to 1 microM. DNA synthesis in the growth phase and confluency was stimulated within 10 h after addition of 0.1 microM retinoic acid. [3H]Retinoic acid binding, which was inhibited by simultaneous addition of excess unlabeled retinoic acid, was detected in both the cytosolic and nuclear fractions of the chondrocytes. The retinoic acid binding capacity of the nuclear fraction was increased by pretreating the cells with retinoic acid. These results indicate that retinoic acid binds to both the cytosolic and nuclear fractions of cultured chondrocytes, and induces their proliferation and dedifferentiation.     

High-dose retinoic acid modulates rat calvarial osteoblast biology

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.     

Retinoic acid-induced alterations of growth and morphology in an established epithelial line

Retinoic acid induced alterations in cellular growth, morphology, and adhesion in a human intestinal epithelial line. Inhibition of growth was dependent on cell density, with the greatest inhibition of growth observed at low cell density. As cells approached confluence, the extent of inhibition was diminished. Cells grown in the presence of retinoic acid appeared elongated and showed enhanced detachability with trypsin. Control cells grew in tight clusters with maximum cell-cell contact. This is in contrast to retinoic acid-treated cells which did not grow in clusters. In addition, it was observed that retinoic acid inhibited glycosaminoglycan biosynthesis. These observations are uniquely different from those reported for non-epithelial cells.     

Purification and properties of retinoic acid-binding protein from chick-embryo skin.

Retinoic acid-binding protein, which is considered to mediate the biological function of retinoic acid in epithelial differentiation and in the possible control of tumorigenesis, was reproducibly purified from chick-embryo skin by using DEAE-Sephadex and Sephadex G-100 column chromatography and isoelectric focusing. About 1mg of protein was isolated from 60g of skin. The purified protein-ligand complex was found to be homogeneous by electrophoresis on polyacrylamide gels. The binding protein has mol.wt. 17800 and pI 4.5. The binding of [3H]retinoic acid to the protein was completely inhibited by mercury compounds. The inhibition is reversible on treatment without dithithreitol; about 50% of the retinoic acid-binding capacity of the mercury-compound-treated protein is restored by chromatography on Sephadex G-25. iodoacetamide treatment of the protein irreversibly inhibits about 50% of retinoic acid binding.     

PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid

Retinoic acid stimulates α-secretase processing of amyloid precursor protein (APP) and decreases β-secretase cleavage that leads to amyloid-β formation. Here, we investigated the effect of retinoic acid on the two putative α-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the β-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of α-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.     

Assessment of retinoid-induced differentiation of F9 embryonal carcinoma cells with an enzyme-linked immunoadsorbent assay for laminin: Statistical comparison of dose-response curves

A convenient procedure, using enzyme-linked immunoadsorbent assay of laminin, to measure retinoid-induced F9-cell differentiation into parietal endoderm was developed. Dose-response curves were fitted with the Allfit program, a statistical method for the analysis and simultaneous comparison of sigmoidal curves, which has been modified for use with a microcomputer. The procedure was standardized with respect to time of retinoid incubation, time-course of laminin production, effects of dibutyryl cAMP, and nature of individual dose-response curves. Retinoic acid produced a half-maximal response at 1.3 nM. Retinol was 175-fold less potent than retinoic acid and required 72 h to effect a maximum response, in contrast to 48 h for retinoic acid. Six oxidized and/or isomerized metabolites of retinoic acid, including 13-cis-retinoic acid, were less potent than retinoic acid, but were more potent than retinol. The dose-response curves had identical slopes with the exception of those obtained with 13-cis-4-oxo- and 4-oxo-16-hydroxyretinoic acids, the only metabolites tested with two structural alterations relative to retinoic acid. Multiple functional group alterations were synergistic in deactivating retinoic acid. The synthetic retinoids 13-cis-N-ethylretinamide and 4-hydroxyphenylretinamide and the steroid hormone 1,25-dihydroxycholecalciferol were inactive.     

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD+ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.     

Effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor

In this study the effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor (EGF) in mouse fibroblast Balb/c 3T6 cells are further examined. Retinoic acid treatment of 3T6 cells results in a sixfold enhancement of 125I-labeled mouse EGF binding when assayed at 37 degrees C. In both retinoic acid-treated and control cells, cell-associated 125I-EGF is rapidly internalized, degraded, and secreted. Retinoic acid treatment does not seem to have a significant effect on the rate of internalization and degradation of EGF. At 0 degrees C, internalization of EGF is strongly inhibited in both retinoic acid-treated and control cells. Under these conditions retinoic acid-treated cells still exhibit a tenfold higher level of EGF binding compared to control cells. When exposed to high concentrations of EGF both retinoic acid-treated and control cells "down-regulate" their EGF receptors. And although the growth rate of retinoic acid-treated cells is about half that of control cells, the rate at which EGF binding capacity is restored after down-regulation is about three times as fast as in control cells. No direct antagonism on EGF binding was observed between the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid. EGF is a potent mitogen for 3T6 cells in serum-free medium; retinoic acid inhibits the mitogenic activity of EGF even though it increases EGF binding. Retinoic acid also inhibits cell proliferation induced by sarcoma growth factor (SGF) and insulin.     

Retinoic acid prevents Chlamydia pneumoniae-induced foam cell development in a mouse model of atherosclerosis

Chlamydia pneumoniae, a common respiratory pathogen, has been associated with cardiovascular disease. C. pneumoniae infection accelerates atherosclerotic lesion development in hyperlipidemic animals. Retinoic acid, an anti-oxidant, inhibits infection of endothelial cells by C. pneumoniae. The present study demonstrated that retinoic acid suppresses the acceleration of foam cell lesion development induced by C. pneumoniae in hyperlipidemic C57BL/6J mice. Retinoic acid treatment had no effect on foam cell lesion development in uninfected animals. Lung infection and duration was decreased in treated mice, suggesting one mechanism by which retinoic acid reduces C. pneumoniae-accelerated foam cell lesion formation in hyperlipidemic mice.     

Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.