Biological activity of all-trans retinol requires metabolic conversion to all-trans retinoic acid and is mediated through activation of nuclear retinoid receptors in human keratinocytes

S B Kurlandsky,J H Xiao,E A Duell,J J Voorhees,G J Fisher

doi:10.1016/s0021-9258(20)30065-x

S B Kurlandsky, J H Xiao + Show 3 more

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https://doi.org/10.1016/s0021-9258(20)30065-x

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Abstract

The biological activity of all-trans retinol, in human keratinocytes, was investigated through metabolic and functional analyses that assessed the capacity for retinol uptake and metabolism and the mechanism of retinol-induced activation of gene transcription. Human keratinocytes converted all-trans retinol predominantly to retinyl esters, which accounted for 60 and 90% of cell-associated radiolabel after a 90-min pulse and a 48-h chase, respectively. Human keratinocytes also metabolized all-trans retinol to low levels of all-trans retinoic acid (11.47-131.3 ng/mg of protein) in a dose-dependent manner, between 0.3 and 10 microM added retinol. Small amounts of 13-cis retinoic acid (5.47-8.62 ng/mg of protein) were detected, but 9-cis retinoic acid was detected only when keratinocytes were incubated with radiolabeled retinol. There was no accumulation of the oxidized catabolic metabolites 4-hydroxy- or 4-oxoretinoic acid; however, 5,6-epoxy retinoic acid was detected at pharmacological levels (10 and 30 microM) of added retinol. Biological activity of retinol was assessed through analysis of two known retinoic acid-mediated responses: 1) reduction of type I epidermal transglutaminase and 2) activation of a retinoic acid receptor-dependent reporter gene, beta RARE3-tk-CAT. Both all-trans retinol and all-trans retinoic acid reduced type I epidermal transglutaminase in a dose-dependent manner; however, the ED50 for all-trans retinol (10 nM) was 10 times greater than for all-trans retinoic acid (1 nM). All-trans retinol also stimulated beta RARE3-tk-CAT reporter gene activity in a dose-dependent manner. Half-maximal induction was observed at 30 nM retinol, which was again 10-fold greater than observed with all-trans retinoic acid. Cotransfection of human keratinocytes with expression vectors for dominant negative mutant retinoic acid and retinoid X receptors reduced retinol-induced beta RARE3-tk-CAT reporter gene activation by 80%. Inhibition of conversion of all-trans retinol or all-trans retinaldehyde to all-trans retinoic acid by citral reduced beta RARE3-tk-CAT activity 98 and 86%, respectively. These data demonstrate that retinol-induced responses in human keratinocytes are mediated by its tightly regulated conversion to retinoic acid, which functions as a ligand to activate nuclear retinoic acid receptors.

Highlights

Levels in Cultured HumanKeratinocytes by All-trans Retinol-Having demonstrated thatproliferating human keratinocytes convert all-trans retinol to all-trans retinoic acid, we examined the biological effectiveness of physiological levels of
The levels of all-trans retinoic acid formed wererelatively low, the estimated concentrations achieved would be expectedto be capable of activating nuclear retinoic acid receptors [39, 51, 52]
Evidence indicates that holo CRBP is a substrate for both retinyl ester formation [3] and retinol oxidation [58] and that apoCRBPinhibits 1ecithin:retinol acyltransferase and stimulates retinyl ester hydrolysis [59]

Summary

EXPERIMENTAL PROCEDURES

Keratinocyte cultures were treated at 90% confluence for analysis of retinol nol, extraction recovery was monitored by addition of PHIretinoic acid to cell suspensions. Nuclear napping of All-trans RetinoicAcid Formed from All-trans pended in PBSfor determination of total cell-associated radioactivitbyy retinol-Cultures of human keratinocytes were treated wLit3hH1retinol liquid scintillation. The dose dependence of all-trans retinol uptake and metabolism was lowingincubation,extractswereappliedtoaPD-lOcolumn,andCRABPII studied by incubating keratinocyte cultures with varying amounts (0.7 was eluted in tvhoeid volume as described by Levin et al(.12).Fractions nM to 30 p ~of) [3Hlretinolfor a single time period of 20 min. Protein con- Leuels-Human keratinocytes, seeded into96-well culture plates, were centrations were determinedby Coomassie Blue stainingof aliquots of treated for 4 days with all-trans retinol, or all-trans retinoic acid, in cell suspensions [36]

IOOOX stock solutions in ethanoUBHT a t concentrations ranging from

AND DISCUSSION

CultureHd uman

EXPRESSION VECTOR

Retinol Activates Gene lFanscription in Human Keratinocytes