Joseph L. Napoli

Retinoic acid: its biosynthesis and metabolism.

This article presents a model that integrates the functions of retinoid-binding proteins with retinoid metabolism. One of these proteins, the widely expressed (throughout retinoid target tissues and in all vertebrates) and highly conserved cellular retinol-binding protein (CRBP), sequesters retinol in an internal binding pocket that segregates it from the intracellular milieu. The CRBP-retinol complex appears to be the quantitatively major form of retinol in vivo, and may protect the promiscuous substrate from nonenzymatic degradation and/or non-specific enzymes. For example, at least seven types of dehydrogenases catalyze retinal synthesis from unbound retinol in vitro (NAD+ vs. NADP+ dependent, cytosolic vs. microsomal, short-chain dehydrogenases/reductases vs. medium-chain alcohol dehydrogenases). But only a fraction of these (some of the short-chain de-hydrogenases/reductases) have the fascinating additional ability of catalyzing retinal synthesis from CRBP-bound retinol as well. Similarly, CRBP and/or other retinoid-binding proteins function in the synthesis of retinal esters, the reduction of retinal generated from intestinal beta-carotene metabolism, and retinoic acid metabolism. The discussion details the evidence supporting an integrated model of retinoid-binding protein/metabolism. Also addressed are retinoid-androgen interactions and evidence incompatible with ethanol causing fetal alcohol syndrome by competing directly with retinol dehydrogenation to impair retinoic acid biosynthesis.

Cellular Retinol-binding Protein-supported Retinoic Acid Synthesis RELATIVE ROLES OF MICROSOMES AND CYTOSOL

This study shows that microsomal retinol dehydrogenases, versus cytosolic retinol dehydrogenases, provide the quantitatively major share of retinal for retinoic acid (RA) biogenesis in rat tissues from the predominant substrate available physiologically, holo-cellular retinol-binding protein, type I (CRBP). With holo-CRBP as substrate in the absence of apo-CRBP microsomal retinol dehydrogenases have the higher specific activity and capacity to generate retinal used for RA synthesis by cytosolic retinal dehydrogenases. In the presence of apo-CRBP, a potent inhibitor of cytosolic retinol dehydrogenases (IC = 1 μM), liver microsomes provide 93% of the total retinal synthesized in a combination of microsomes and cytosol. Cytosolic retinol dehydrogenase(s) and the isozymes of alcohol dehydrogenase expressed in rat liver had distinct enzymatic properties; yet ethanol inhibited cytosolic retinol dehydrogenase(s) (IC = 20 μM) while stimulating RA synthesis in a combination of microsomes and cytosol. At least two discrete forms of cytosolic retinol dehydrogenase were observed: NAD- and NADP-dependent forms. Multiple retinal dehydrogenases also were observed and were inhibited partially by apo-CRBP. These results provide new insights into pathways of RA biogenesis and provide further evidence that they consist of multiple enzymes that recognize both liganded and nonliganded states of CRBP.

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 alcohol 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 Km substrate.

cDNA cloning and characterization of a cis-retinol/3alpha-hydroxysterol short-chain dehydrogenase.

We report a mouse cDNA that encodes a 317-amino acid short-chain dehydrogenase which recognizes as substrates 9-cis-retinol, 11-cis-retinol, 5α-androstan-3α,17β-diol, and 5α-androstan-3α-ol-17-one. Thiscis-retinol/androgen dehydrogenase (CRAD) shares closest amino acid similarity with mouse retinol dehydrogenase isozymes types 1 and 2 (86 and 91%, respectively). Recombinant CRAD uses NAD+ as its preferred cofactor and exhibits cooperative kinetics for cis-retinoids, but Michaelis-Menten kinetics for 3α-hydroxysterols. Unlike recombinant retinol dehydrogenase isozymes, recombinant CRAD was inhibited by 4-methylpyrazole, was not stimulated by ethanol, and did not require phosphatidylcholine for optimal activity. CRAD mRNA was expressed intensely in kidney and liver, in contrast to retinol dehydrogenase isozymes, which show strong mRNA expression only in liver. CRAD mRNA expression was widespread (relative abundance): kidney (100) > liver (92) > small intestine (9) = heart (9) > retinal pigment epithelium and sclera (4.5) > brain (2) > retina and vitreous (1.6) > spleen (0.7) > testis (0.6) > lung (0.4). CRAD may catalyze the first step in an enzymatic pathway from 9-cis-retinol to generate the retinoid X receptor ligand 9-cis-retinoic acid and/or may regenerate dihydrotestosterone from its catabolite 5α-androstan-3α,17β-diol. These data also illustrate the multifunctional nature of short-chain dehydrogenases and provide a potential mechanism for androgen-retinoid interactions.

Cloning of a rat cDNA encoding retinal dehydrogenase isozyme type I and its expression in E. coli

Peptides sequenced from the purified rat liver cytosolic retinal dehydrogenase P1 [Posch, K.C., Burns, R.D. and Napoli, J.L., 1992. Biosynthesis of all-trans-retinoic acid from retinal: recognition of retinal bound to cellular retinol-binding protein (type I) as substrate by a purified cytosolic dehydrogenase. J. Biol. Chem. 267, 19676-19682] were used to design oligonucleotides for cloning its cDNA. The deduced amino acid sequence of P1, now designated retinal dehydrogenase type I or RalDH(I), has close similarity with mouse AHD-2 and rat kidney aldehyde dehydrogenase, but is distinct from rat phenobarbital-inducible aldehyde dehydrogenase (PIADH), the presumed rat liver homolog of mouse AHD-2. Rat kidney (100%) and lung (88%) show relatively high mRNA levels of RalDH(I), liver (34%) and brain (22%) have moderate levels, and testis (8%) has low levels. Retinoid status affects RalDH(I) mRNA levels differently in different tissues. E. coli-expressed RalDH(I) exhibits allosteric kinetics for retinal with a Hill coefficient of 1.7, a K0.5 value of 1.4 microM and a Vmax of 52 nmol min(-1) mg(-1) protein. These data establish the cospecificity of P1 and RalDH(I), show that retinoid status affects expression of its mRNA in a tissue-dependent manner, and illustrate that aldehyde dehydrogenase isozymes with extensive homology can participate in different metabolic paths, e.g., RalDH vs. PIADH.

Coexpression of the mRNAs encoding retinol dehydrogenase isozymes and cellular retinol‐binding protein

We used in situ hybridization of adult rat tissue to show that mRNAs encoding cellular retinol‐binding protein (CRBP) and retinol dehydrogenase (RoDH) isozymes I/III and II were expressed in hepatocytes uniformly throughout the liver lobule, but were absent from Kupffer cells and endothelial cells of blood vessels and bile ducts. In kidney, CRBP, RoDH(I), and RoDH(II) were found in the proximal tubules of the cortex. Distal tubules, Henles loops, collecting ducts, and glomeruli showed little, if any, expression. In testis, CRBP, RoDH(I), and RoDH(II) were found in Sertoli cells. Expression, albeit weaker, also occurred in spermatogonia and primary spermatocytes. Peritubular cells and other germ cells had even weaker expression. Only CRBP and RoDH(II) mRNA were detected in interstitial cells. In lung CRBP, RoDH(I) and RoDH(II) were expressed most intensely in the epithelium of the bronchi and bronchioli, but also occurred in the simple columnar epithelial cells of the alveolar duct and in alveolar type II cells. These data are consistent with the hypothesis that holo‐CRBP serves as substrate for retinoic acid biosynthesis because they show that the substrate and the enzyme occur in the same cellular loci in vivo. These data also indicate that multiple cellular sites of retinoic acid biosynthesis occur throughout tissues. Also, the general concordance between mRNA localization and CRBP expression patterns, revealed by previous immunocytochemistry studies, supports and extends the conclusion that CRBP mRNA expression correlates with CRBP expression, based earlier on comparing RNA assays with radioimmunoassays. J. Cell. Physiol. 173:36–43, 1997.

Enzymatic characteristics of retinal dehydrogenase type I expressed in Escherichia coli

We expressed RalDH(I) in Escherichia coli and have shown that it functions in vitro with the complex CRBP-retinal (cellular retinol-binding protein) as substrate, either generated in situ from the complex CRBP-retinol and microsomal retinol dehydrogenases or provided directly as CRBP-retinal. Recombinant RalDH(I) had kinetic constants with CRBP-retinal of: Hill coefficient 1.8; K0.5 0.8 microM; and Vm 1.5 nmol/min/mg of protein at 25 degrees C. Apo-CRBP inhibited the reaction with CRBP-retinal with an IC50 of 1.4 microM. Citral inhibited RalDH(I) with an IC50 of approximately 1 microM compared to an IC50 of approximately 12 microM for RalDH(II), but did not serve as substrate for RalDH(I). RalDH(I) did not catalyze efficiently the dehydrogenation of acetaldehyde, but showed higher Vmax/Km values for hexanal, octanal, decanal and benzaldehyde than for either propanal or retinal. These data extend the characterization of RalDH(I), show that apo-CRBP competes with holo-CRBP as substrate for RalDH(I), and expand insight into the pathways of retinoic acid biogenesis from the most abundant substrates in vivo, retinoid-liganded CRBP.

Microsomes convert retinol and retinal into retinoic acid and interference in the conversions catalyzed by cytosol

Rat liver microsomes converted retinol into retinal and retinoic acid. The production of retinal was observed over a range of substrate concentrations (10-100 microM), but retinoic acid was detected only at retinol concentrations of 50 microM or higher. At 50 microM retinol, the rate of microsomal retinal production was 2-fold greater than that of cytosol, but the rate of retinoic acid synthesis was 4-fold less than that of cytosol. Retinal was also converted into retinoic acid by rat liver microsomes, but at a rate 2-5% of that catalyzed by cytosol. Microsomes also interfered with the conversion of retinol and retinal into retinoic acid by rat liver cytosol. A 50% decrease in the cytosolic rates of retinoic acid production from retinol or retinal was caused by microsomal to cytosolic protein ratios of 0.1 and 0.5, respectively. Under the incubation conditions, which included NAD in the medium, addition of microsomes to cytosol did not affect the elimination half-life of retinol or retinoic acid, but did decrease the elimination half-life of retinal by 2-fold. These data show that retinal synthesis from retinol does not necessarily reflect retinoic acid synthesis and suggest that liver microsomes sequester free retinol and convert it into retinal primarily for elimination, rather than to serve as substrate for cytosolic retinoic acid synthesis.

Microsomal retinal synthesis: retinol vs. holo-CRBP as substrate and evaluation of NADP, NAD and NADPH as cofactors

Holo-CRBP (cellular retinol binding protein) is recognized specifically by an NADP-dependent microsomal retinol dehydrogenase and protects retinol from conversion into retinal by NAD and NADPH dependent dehydrogenases. The synthesis of retinal from free retinol is catalyzed by both NADP- and NAD-dependent pathways, with the former being the preferred one (Km of 4 vs. 22 microM for retinol, and Vmax/Km of 33 vs. 9, respectively). NADPH does not support quantitatively significant retinal synthesis from physiological concentrations of retinol or holo-CRBP, if an NADPH regenerating system is used to prevent NADP formation.

Increase in β‐1,4‐Galactosyltransferase Activity During PC12 Cell Differentiation Induced by Forskolin and 2‐Chloroadenosine

Galactosyltransferase (GAL Tase) activity was measured in differentiating PC12 cells induced by either forskolin or 2‐chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2‐chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2‐chtoroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2‐chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)‐ and protein kinase A‐dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine F9 carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.