Cloning and Characterization of Retinol Dehydrogenase Transcripts Expressed in Human Epidermal Keratinocytes

Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3␣-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis.All-trans-retinoic acid is a small lipophilic molecule derived from vitamin A that regulates the expression of Ͼ530 different genes in various types of cells and tissues through binding to nuclear transcription factors (retinoic acid receptors (RARs)) 3 (1). Skin is one of the best characterized targets of retinoid action (2, 3) and contains all of the major components of retinoid signaling and metabolic machinery such as retinoid receptors RAR␥ and RXR␣ (4); cellular retinoic acid binding protein (5) and cellular retinol binding protein (6, 7); retinaldehyde dehydrogenase (8); lecithin retinol acyltransferase (9); and cytochrome P450 (10). Keratinocytes can synthesize retinoic acid in situ from plasma-derived retinol (11-13); the reported concentration of retinoic acid in the epidermal cells is very low (Յ20 nM), and it is controlled strictly (14). Concentrations that exceed the optimal range suppress differentiation and promote hyperproliferation, whereas concentrations below this range lead to formation of orthokeratotic epithelium (15).Retinoic acid is synthesized from retinol in two steps; first, retinol is reversibly oxidized to retinaldehyde, and then retinaldehyde is oxidized irreversibly to retinoic acid. The oxidation of retinol to retinaldehyde is the rate-limiting step in retinoic ac...

mentioning

confidence: 66%

Section: Expression Of Human Sdrs In Transgenic Skinmentioning

confidence: 99%

Retinol Dehydrogenase 10 but Not Retinol/Sterol Dehydrogenase(s) Regulates the Expression of Retinoic Acid-responsive Genes in Human Transgenic Skin Raft Culture

Lee¹,

Belyaeva²,

et al. 2011

“…1). Mutants RD (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) or RD(1-30), which lack the first 18 and 30 amino acid residues of Rdh1, respectively, did not localize to the ER. Surprisingly both distributed in the mitochondria fraction with cytochrome c and localized in mitochondria in intact cells (Figs.…”

Section: Resultsmentioning

confidence: 99%

Elements in the N-terminal Signaling Sequence That Determine Cytosolic Topology of Short-chain Dehydrogenases/Reductases

Zhang

Napoli

2004

Self Cite

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11␤-hydroxysteroid dehydrogenase (11␤-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/ GFP, Crad1(1-22)/GFP, and 11␤-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N-and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289 -311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.Retinol requires conversion into all-trans-retinoic acid to fulfill the vitamin A function of regulating gene expression that begins shortly after conception and continues throughout vertebrate life (1, 2). Retinoid-active SDRs 1 recognize the major physiological form of retinol, retinol bound with CRBP, to generate retinal for irreversible conversion into all-trans-retinoic acid (3). The most active of the retinol dehydrogenases include mouse Rdh1 and its orthologs, rat Rodh1 and -2 and human RDH-E/RoDH4 (4 -8). rRodh2 e.g. has lower apparent K m values with holo-CRBP than with unbound retinol and catalyzes retinal synthesis in the presence of excess apoCRBP. Dehydrogenation of retinol in the presence of excess CRBP with concentrations of both ligand and binding protein in the micromolar range indicates that the reaction proceeds through the SDR accessing CRBP-bound retinol because the CRBP-retinol complex has a K d value ϳ0

“…However, the level of RA in the plasma is around 4 -14 nM (14,15), which may be insufficient to supply the needed cellular RA level for its biological activity in vitamin A target tissues and cells. In addition, in some vitamin A target tissues and cells, such as skin and keratinocytes (16), the access to blood vessels can be quite limited. Thus, there is a need in these vitamin A target cells for an efficient machinery to transport and to metabolize plasma retinol into various RA metabolites.…”

mentioning

confidence: 99%

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Soref¹,

Di²,

Hayden

et al. 2001